Titin Isoform Research Articles

A Novel Role for PP5 in Regulating Titin Phosphorylation and Function in the Heart

Within the N2B/N2BA isoforms of the giant protein titin, a cardiac-specific unique sequence (N2Bus) can be phosphorylated by various protein kinases, including PKA, PKG, ERK2, and CaMKII, which reduces titin-based stiffness. Failing human hearts are hypo-phosphorylated at the N2Bus. Specific phosphatases acting on N2Bus are not known. We carried out a yeast-2-hybrid screen using human N2Bus (“bait”) and a human cardiac cDNA library (“prey”) and found the catalytic domain of protein-phosphatase-5 (PP5c) as a binding partner of N2Bus. The interaction was verified by GST-pulldown assays. PP5 is unique within the family of serine/threonine protein phosphatases because of the presence of tetratricopeptide repeats (TPR) at the molecule's N-terminus. PP5 is well expressed in human and mouse hearts and elevated in failing hearts. PP5 is autoinhibited due to interaction of the TPR domain with the catalytic C-terminus. However, PP5 can be activated by proteolytic cleavage of the TPR region, through binding of the TPR domain by arachidonic acid, or via binding of heat-shock-protein-90 (HSP90). In PP5-overexpressing transgenic (TG) mouse hearts, we found PP5 to co-localize with the titin-N2Bus in the sarcomeric I-band, as shown by immunofluorescence and immunoelectron microscopy. Autoradiography/back-phosphorylation assays and the detection of N2Bus-phosphosites by phospho-specific anti-titin antibodies showed that recombinant PP5 dephosphorylates recombinant human N2Bus previously phosphorylated by ERK2, PKA, or PKG. PP5 also dephosphorylates the cardiac titin isoforms, N2B and N2BA, in human heart tissue. PP5 TG mouse hearts showed reduced N2Bus phosphorylation compared to wildtype (WT) hearts. Force measurements on permeabilized single cardiomyocytes revealed increased passive stiffness in PP5 TG vs. WT hearts. To conclude, PP5 binds to titin N2Bus and acts as an antagonist to the protein-kinase-mediated effects on titin stiffness. Inhibition of the PP5 effect on titin might benefit stiff hearts.

Impact of cGMP-PKG Pathway Modulation on Titin Phosphorylation and Titin-Based Myocardial Passive Stiffness

RATIONALE: The crucial contribution of the giant myofilament protein titin to diastolic stiffness and cardiomyocyte passive force(Fpassive) is dependent, in part, on titin isoform composition and phosphorylation. Phosphorylation of titin by cyclic guanosine monophosphate(cGMP)-dependent protein kinase G(PKG) lowers titin-based stiffness, thus mediating a mechanical signaling process that is disturbed in heart failure. OBJECTIVE: To elucidate which elements of the nitric oxide (NO) cGMP-PKG signaling network are critical for titin phosphorylation and stiffness in vivo. METHODS AND RESULTS: We employed genetic knockout(KO) mouse models deficient for cGMP-PKG pathway enzymes, including cardiomyocyte-specific deletion of the guanylyl cyclase(GC)-A receptor and cGMP-dependent-PKG(cGKI), and global deletion of soluble GC(sGC). We assessed titin phosphorylation and Fpassive of single permeabilized cardiomyocytes recorded before/after PKG administration. In all three models, all-titin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within titin-N2-Bus was hypophosphorylated in all three KO-models. Unexpectedly, mass spectrometry analysis revealed that most class-1 titin phospho-sites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class-1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG; indeed, this was associated with upregulation of several kinases that phosphorylate titin and a clear rise in Fpassive in KO vs. WT cardiomyocytes. While administration of PKG lowered Fpassive of WT and KO cardiomyocytes in all models, this effect was more pronounced in the cGKI KO. CONCLUSIONS: Multiple in vivo phosphorylated class I titin phospho-sites were identified within the molecular spring segment, some of which depended on the cGMP-PKG pathway. While cGMP-activated PKG remains an important titin-targeting kinase, many titin phospho-sites may be regulated through a network of protein kinases/phosphatases.

Exercise Mitigates Increased Diastolic Stiffness in the Titin IA KO Mouse

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.

The PEVK Region of Titin: Deciphering the Molecular Interactions of Elasticity

Titin is an elastic protein which spans half a sarcomere in muscles. Active muscles contract using the movement of thin and thick filaments in a well established way, but not all types of contractions can be explained with existing models. Titin is known to play a role in certain types of contractions but this role is not well understood. There is a variety of titin isoforms, but there are two conserved types of domains in all isoforms: immunoglobulin repeats and the PEVK region. Previous studies have shown that titin elongates first by separating immunoglobulin domains followed by stretching of the PEVK region (which requires higher forces). The PEVK is a highly elastic region of titin with two different sequence patterns; lysine rich PPAK (positively charged) and glutamic acid rich poly-E (negatively charged). The goal of this project is to characterize the molecular interactions within the PEVK region, using human titin as a model, since these interactions towards titin's elasticity have not been clearly elucidated. Our initial work has been to characterize the interactions between single PPAK and poly-E sequences. Preliminary circular dichroism data of the PPAK and poly-E show a random coil structure, consistant with the literature. Circular dichroism spectra for PPAK and poly-E mixtures deviates from the additive spectra of the individual peptides under physiological ionic strengths, suggesting an interaction between PPAK and poly-E. We are now expanding these studies to include more complex sequences that contain varying number of PPAK and poly-E segments in the same peptide and using these constructs quantitate the interactions. These studies will help us to understand the molecular nature of PEVK region and the forces contributing for its elasticity.

Effect of Plyometrics on the Energy Cost of Running and MHC and Titin Isoforms.

Several training strategies such as plyometrics have been shown to improve running economy; however, its physiological basis remains elusive. To examine the effect of plyometric training on the energy cost of running (ECR, J · kg(-1) · min(-1)), titin, and myosin heavy chain (MHC) isoforms. Subjects were randomly assigned to a 6-wk plyometric treatment (P; n = 11) or control group (C; n = 11). Preintervention and postintervention outcomes included body composition, vertical jump, sit-and-reach, maximal oxygen consumption (VO2max), speed at onset of blood lactate, 3-km time trial performance, ECR, and a vastus lateralis muscle biopsy for protein analysis. Plyometric intervention resulted in improved time trial (P, 2.6% faster, P = 0.04; C, 1.6%, P = 0.17). VO2max improved in the P group (5.2%, P = 0.03), whereas the C group increased by 3.1% (P = 0.20). The ECR decreased in the P group as the result of 6 wk of plyometric training (P = 0.02 for stage 3), whereas it increased in the C group (P = 0.02 for stage 3). The ECR correlated strongly with performance at stages 2, 3, and 4 (r > 0.8, P < 0.001) independent of group. There was no significant main effect of group, time, or interaction on any of the protein isoforms analyzed. A negative correlation was found between the ECR at stage 7 and MHC IIa (r = -0.96, P < 0.001), and the ECR at stage 6 with titin isoform 1 (T1)/T2 ratio (r = -0.69, P = 0.007) independent of group. Six weeks of plyometric training improved running performance and the ECR despite no measurable changes in MHC and titin isoforms. However, higher MHC IIa and lower T1/T2 isoform ratios correlated to lower ECR.

Eccentric contraction: unraveling mechanisms of force enhancement and energy conservation.

During the past century, physiologists have made steady progress in elucidating the molecular mechanisms of muscle contraction. However, this progress has so far failed to definitively explain the high force and low energy cost of eccentric muscle contraction. Hypotheses that have been proposed to explain increased muscle force during active stretch include cross-bridge mechanisms, sarcomere and half-sarcomere length non-uniformity, and engagement of a structural element upon muscle activation. The available evidence suggests that force enhancement results from an interaction between an elastic element in muscle sarcomeres, which is engaged upon activation, and the cross-bridges, which interact with the elastic elements to regulate their length and stiffness. Similarities between titin-based residual force enhancement in vertebrate muscle and twitchin-based 'catch' in invertebrate muscle suggest evolutionary homology. The winding filament hypothesis suggests plausible molecular mechanisms for effects of both Ca(2+) influx and cross-bridge cycling on titin in active muscle. This hypothesis proposes that the N2A region of titin binds to actin upon Ca(2+) influx, and that the PEVK region of titin winds on the thin filaments during force development because the cross-bridges not only translate but also rotate the thin filaments. Simulations demonstrate that a muscle model based on the winding filament hypothesis can predict residual force enhancement on the descending limb of the length-tension curve in muscles during eccentric contraction. A kinematic model of titin winding based on sarcomere geometry makes testable predictions about titin isoforms in different muscles. Ongoing research is aimed at testing these predictions and elucidating the biochemistry of the underlying protein interactions.

A porcine model of hypertensive cardiomyopathy: implications for heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFPEF) evolves with the accumulation of risk factors. Relevant animal models to identify potential therapeutic targets and to test novel therapies for HFPEF are missing. We induced hypertension and hyperlipidemia in landrace pigs (n = 8) by deoxycorticosteroneacetate (DOCA, 100 mg/kg, 90-day-release subcutaneous depot) and a Western diet (WD) containing high amounts of salt, fat, cholesterol, and sugar for 12 wk. Compared with weight-matched controls (n = 8), DOCA/WD-treated pigs showed left ventricular (LV) concentric hypertrophy and left atrial dilatation in the absence of significant changes in LV ejection fraction or symptoms of heart failure at rest. The LV end-diastolic pressure-volume relationship was markedly shifted leftward. During simultaneous right atrial pacing and dobutamine infusion, cardiac output reserve and LV peak inflow velocities were lower in DOCA/WD-treated pigs at higher LV end-diastolic pressures. In LV biopsies, we observed myocyte hypertrophy, a shift toward the stiffer titin isoform N2B, and reduced total titin phosphorylation. LV superoxide production was increased, in part attributable to nitric oxide synthase (NOS) uncoupling, whereas AKT and NOS isoform expression and phosphorylation were unchanged. In conclusion, we developed a large-animal model in which loss of LV capacitance was associated with a titin isoform shift and dysfunctional NOS, in the presence of preserved LV ejection fraction. Our findings identify potential targets for the treatment of HFPEF in a relevant large-animal model.

WITHDRAWN: Titin Isoform Expression in Aortic Stenosis and Aortic Regurgitation: Differences and Relationships with Functional and Geometric Characteristics

WITHDRAWN: Titin Isoform Expression in Aortic Stenosis and Aortic Regurgitation: Differences and Relationships with Functional and Geometric Characteristics

197 Left Atrial Remodelling and Atrioventricular Coupling in a Canine Model of Early Heart Failure with Preserved Ejection Fraction

Background Left atrial (LA) compliance and contractility are key determinants of left ventricular (LV) stroke volume. LA remodelling and dysfunction in heart failure with preserved ejection fraction (HFpEF) are considered to be late presenting features, secondary to hypertension-induced LV diastolic dysfunction and chronic LA pressure overload. However, a recent unifying hypothesis has stipulated that systemic and coronary microvascular inflammation drive HFpEF pathophysiology. Considering this paradigm, we hypothesised that diminished LA compliance and contractile function occur early during HFpEF development and further impair cardiac performance. Methods Cardiac magnetic resonance imaging, echocardiography, invasive LV and LA pressure volume (PV) analysis and tissue analysis were performed in an experimental model of early-stage HFpEF (elderly dogs with renal wrap induced hypertension and exogenous aldosterone; n = 9) and young control dogs (sham surgery; n = 13). Results Early HFpEF was associated with LA enlargement, LA myocyte hypertrophy, and enhanced LA contractile function (LA active emptying fraction 18 ± 6 vs 12 ± 2%, p = 0.03; LA end-systolic PV relationship slope: 2.7 ± 0.9 HFpEF vs 1.5 ± 0.6 mmHg/mL controls, p = 0.01). However left atrioventricular systolic coupling was impaired (ratio of LV to LA end-systolic PV relationship slope 1.3 ± 0.4 HFpEF vs 0.8 ± 0.4 Controls, p 2 , p = 0.07) and inversely correlated with LA s (r=-0.66, p = 0.006). Titin isoform composition (N2BA/N2B ratio) was similar between groups, but titin hyperphosphorylation was seen in HFpEF LA and LV tissue. LA titin hyperphosphorylation correlated with LA s (r = 0.54, p = 0.03). LA microvascular reactivity was also diminished in HFpEF versus controls. Conclusions Maladaptive LA remodelling is present in early-stage experimental HFpEF and compromises overall cardiac performance. Reduced LA compliance in HFpEF may relate to contributions from LA ischaemia and titin-based cardiomyocyte stiffening. Thus, pathophysiological mechanisms involved in the development of HFpEF directly affect both atrial and ventricular myocardium.

TU-F-CAMPUS-I-03: Quantitative Cardiac MRI Reveals Functional Abnormalities in Intrauterine Growth Restricted (IUGR) Baboons

Purpose: Developmental programming occurs in utero, when epigenetic adaptations Result in offspring phenotypes that lead to health consequences persisting throughout life. Programming of cardiac health, due to prenatal malnutrition, has been reported in rodents but not in primate species. Our aim was to determine IUGR is associated with impaired cardiac function in later life in a baboon model. Methods: Intrauterine growth restricted (IUGR, mothers were subjected to a 30% calories reduction) baboons were scanned with cine cardiac MRI measuring ejection fraction, left ventricular (LV) volumes, LV peak filling rate (LVPFR) and the LV 3D sphericity index (3DSI). Baboons were anesthetized and the respirator was turned off during scans. Three groups of baboons were studied: adult control (CTR): N=21 (11M, 10 F), age = 5.9±1.3 yr., IUGR: N=9(4M, 5F), age = 4.6±0.5 yr., and normal elderly adults (ELD): N=11(5M, 6 F), age = 15.3±2.4 yr. Images were analyzed using CMR42 software. LV end-diastolic volume (EDV) and end-systolic volumes (ESV) were referenced to body-surface areas (BSA). Linear regression, correlation and ANOVA with Bonferroni correction were performed between for each of the three groups (p<.05 deemed significant). Results: Systolic LV function in all three groups was normal. There were significant differences between the three groups for 3DSI and LVPFR by ANOVA. (p<.01) Linear regression and correlation were performed between 3DSI and LVFR data for each of the three groups. CTR: r=0.01(p=NS); IUGR: r=0.881 (p<.02); ELD: r=0.724 (p<.05). Conclusions: All subjects had normal systolic function but diastolic dysfunction was associated with IUGR and aging. Results are consistent with reports of increased fibrosis and changes in the distribution of titin isoforms in IUGR fetal baboon heart tissues. The data suggest that developmental programming, due to IUGR, accelerates processes leading to diastolic heart dysfunction, which is common with aging. GDC:NIH/K25-KDK089012A. AJK:NIH/R25-EB016631 PN: EU/BRAINAGE-IMPACT OF PRENATAL STRESS ON BRAIN AGE(FP7-HEALTH-2011).

Intermittent stretch training of rabbit plantarflexor muscles increases soleus mass and serial sarcomere number.

In humans, enhanced joint range of motion is observed after static stretch training and results either from an increased stretch tolerance or from a change in the biomechanical properties of the muscle-tendon unit. We investigated the effects of an intermittent stretch training on muscle biomechanical and structural variables. The left plantarflexors muscles of seven anesthetized New Zealand (NZ) White rabbits were passively and statically stretched three times a week for 4 wk, while the corresponding right muscles were used as nonstretched contralateral controls. Before and after the stretching protocol, passive torque produced by the left plantarflexor muscles as a function of the ankle angle was measured. The left and right plantarflexor muscles were harvested from dead rabbits and used to quantify possible changes in muscle structure. Significant mass and serial sarcomere number increases were observed in the stretched soleus but not in the plantaris or medial gastrocnemius. This difference in adaptation between the plantarflexors is thought to be the result of their different fiber type composition and pennation angles. Neither titin isoform nor collagen amount was modified in the stretched compared with the control soleus muscle. Passive torque developed during ankle dorsiflexion was not modified after the stretch training on average, but was decreased in five of the seven experimental rabbits. Thus, an intermittent stretching program similar to those used in humans can produce a change in the muscle structure of NZ White rabbits, which was associated in some rabbits with a change in the biomechanical properties of the muscle-tendon unit.

Insulin and Angiotensin II Regulate Titin Alternative Splicing via Rbm20

ObjectiveThis study is to determine whether titin alternative splicing is regulated by insulin and angiotensin II via a muscle‐specific splicing factor, RNA binding motif 20 (Rbm20).MethodsIn primary cultures, neonatal cardiomyocytes (CMs) were isolated from day 1 rats. CMs from wild type (WT), heterozygous (HT), and homozygous (HM) Rbm20 deficient rats were cultured in serum‐free medium and treated with insulin, insulin plus LY294002 (LY) and angiotensin II for 5 days. In in vivo assay, HT rats were treated with streptozotocin (STZ) for 8 weeks. Titin isoforms were detected by 1% SDS‐agarose gel. Band intensity was quantified with imageJ. Western blot and QRCR were performed for protein and gene expression.ResultsInsulin and angiotensin II increased the expression of smaller titin isoform (N2B) in both WT and HT CMs, but no N2B was rescued by insulin and angiotensin II in HM CMs. The phosphorylation of Akt was increased in WT, HT and HM CMs. LY can inhibit Akt phosphorylation and titin isoform transition in both WT and HT insulin treated CMs. STZ treatment decreased the expression of smaller titin N2B in HT rats. The mRNA levels of titin isoforms were consistent with protein level. Rbm20 expression was increased in insulin treated CMs.ConclusionTitin alternative splicing has been shown to be largely responsible for the diastolic properties of the heart. Rbm20 is the major regulator of the titin alternative splicing. These results indicated that insulin and angiotensin II regulated titin alternative splicing is Rbm20 dependent, and Akt phosphorylation can enhance Rbm20‐regulated titin alternative splicing.Supported by NIH P20 GM103432, University of Wyoming Agricultural Experimental Station and USDA‐NIFA

Seasonal changes in isoform composition of giant proteins of thick and thin filaments and titin (connectin) phosphorylation level in striated muscles of bears (Ursidae, Mammalia).

Seasonal changes in the isoform composition of thick and thin filament proteins (titin, myosin heavy chains (MyHCs), nebulin), as well as in the phosphorylation level of titin in striated muscles of brown bear (Ursus arctos) and hibernating Himalayan black bear (Ursus thibetanus ussuricus) were studied. We found that the changes that lead to skeletal muscle atrophy in bears during hibernation are not accompanied by a decrease in the content of nebulin and intact titin-1 (T1) isoforms. However, a decrease (2.1-3.4-fold) in the content of T2 fragments of titin was observed in bear skeletal muscles (m. gastrocnemius, m. longissimus dorsi, m. biceps) during hibernation. The content of the stiffer N2B titin isoform was observed to increase relative to the content of its more compliant N2BA isoform in the left ventricles of hibernating bears. At the same time, in spite of the absence of decrease in the total content of T1 in the myocardium of hibernating brown bear, the content of T2 fragments decreased ~1.6-fold. The level of titin phosphorylation only slightly increased in the cardiac muscle of hibernating brown bear. In the skeletal muscles of brown bear, the level of titin phosphorylation did not vary between seasons. However, changes in the composition of MyHCs aimed at increasing the content of slow (I) and decreasing the content of fast (IIa) isoforms of this protein during hibernation of brown bear were detected. Content of MyHCs I and IIa in the skeletal muscles of hibernating Himalayan black bear corresponded to that in the skeletal muscles of hibernating brown bear.

Passive Force Analysis of Single Sarcomeres from Muscles Lacking Arginyl-tRNA-Protein Transferase (Ate1)

Introduction: Arginylation is a post-translational process mediated by the enzyme arginyl-tRNA transferase (Ate1). Recent findings from our laboratory suggest an important role of arginylation on the architecture and function of skeletal and cardiac muscles. In this study, we investigated the passive force development of single sarcomeres isolated from soleus muscles of mice lacking Ate1.Methods: Soleus muscles from Ate1 KO and wild type (WT) mice were homogenized to myofibrils. Single sarcomeres in relaxing solution were tested using a pair of micro-needles. The sarcomeres were stretched in steps of 300 nm, starting in a sarcomere length of 2.2μm (±0.2μm). The force produced by the sarcomeres was plotted against the sarcomere length. Mass spectrometry was used to evaluate the presence of arginylation sites on titin. Gel electrophoresis was used to test for isoform modifications due to the lack of Ate1.Results: Single sarcomeres of Ate KO developed less passive force than sarcomere from WT. Mass Spectrometry reviewed 4 sites for arginylation within titin's A-band structure. Electrophoresis gels showed no difference in the titin N2A isoform.Conclusion: Post-translation arginylation of titin A-band does not change titin's isoform, but is required for normal passive force development in soleus muscles.

Analysis of cardiac titin isoform switch around birth.

Analysis of cardiac titin isoform switch around birth.

Abstract 18852: Myofilament Length Dependent Activation: Role of Titin

The cellular basis of the Frank-Starling mechanism is sarcomere length (SL) modulation of myofilament Ca 2+ sensitivity (LDA). The molecular mechanism(s) that underlie LDA are unknown, but recent evidence has implicated the giant protein titin as possible sarcomeric strain sensor responsible for LDA by an, as of yet unidentified, signal transduction pathway. Accordingly, the aim of the present study was to elucidate the impact of SL (from slack=2.0 μm to stretch=2.4 μm) on LDA and sarcomere structure in isolated rat myocardium from either wild-type (WT) or mutant (HM) rats expressing a giant splice isoform of titin. At stretch, WT muscles showed reduced increase in passive tension (-20%) and twitch force (-58%); also, LDA was significantly blunted at the myofilament level. Time-resolved small angle x-ray diffraction of intact twitching muscles during diastole revealed at stretch a significant increase in intensity and spacing of: myosin M2 (+121% &amp; +0.4%) and troponin T3 (+174% &amp; +1.0%); Myosin binding protein C (MyoBPC) also trended to increase (C1%C2). These SL dependent changes in sarcomere structure were absent in HM muscles. Cross-bridge radial spacing (layer line analysis) was significantly reduced at stretch in WT (-8.0%); in HM muscles radial spacing was further, but similarly reduced at both slack and stretch (~-20%). Equatorial spacings and intensity ratios were similar between WT and HM at both slack and stretch. Electron density reconstruction revealed, only in WT, increased mass in both thick and thin filament, and the appearance of an as of yet unidentified moiety spanning the space between the thick and thin filaments at stretch. These results were independently confirmed in skinned myocyte fragments in which endogenous TnC was replaced with fluorescently labelled TnC. Conclusion: Stretch induces structural changes in both thick and thin filaments mediated by titin strain. Moreover, MyoBPC may interact with actin to mediate LDA.

Abstract 17218: Reduced Left Atrial Compliance in Heart Failure With Preserved Ejection Fraction: New Insights Into Pathophysiology

Background: Reduced left atrial (LA) compliance (LAC) contributes to pulmonary venous hypertension in heart failure with preserved ejection fraction (HFpEF); however the underlying mechanisms are poorly defined. We hypothesized that microvascular inflammation with rarefaction and impaired nitric oxide (NO) signaling, LA titin hypophosphorylation and fibrosis may each contribute to reduced LAC in HFpEF. Methods: Invasive LA pressure-volume (PV) analyses (open-chest; pericardiotomy; admittance catheter) were performed in 9 aged dogs with experimental hypertension (bilateral renal wrap + DOCA; HFpEF) compared to 13 sham-operated young adult dogs (control). LAC was measured at end-LA reservoir during acute preload reduction (cava occlusion). LA tissue and LA microvascular function were analyzed. Results: Compared to controls, the curvilinear end-LA reservoir PV relationship ( Fig A ) was shifted upward/leftward in HFpEF (LA diastolic stiffness constant 0.08 vs 0.16 mmHg/mL in controls, p=0.005). LA microvascular density (MVD; CD31 stain) tended to be lower in HFpEF than controls (2154±578 vs 2650±523 vessels/mm 2 , p=0.07) and LA MVD negatively correlated with the LA diastolic stiffness constant (r=-0.66, p=0.006). LA microvessels from HFpEF dogs showed impaired endothelium-dependent shear stress-mediated dilation compared to controls, consistent with impaired NO signaling ( Fig B ). Titin isoform expression was similar between groups, but isoform N2B (stiffer) was (p=0.03) and N2BA (compliant) tended to be (p=0.09) hypophosphorylated in HFpEF LA appendage (LAA) tissue. Fibrosis (Picrosirius Red) was similar between groups in the LA free wall and LAA, but increased in areas adjacent to the pulmonary veins in HFpEF (27±12% vs controls 15±8%, p=0.01). Conclusion: In experimental HFpEF, LAC was reduced in association with evidence of LA microvascular rarefaction and impaired NO signaling, titin hypophosphorylation and regional increases in LA fibrosis.

Increased myocardial stiffness due to cardiac titin isoform switching in a mouse model of volume overload limits eccentric remodeling

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.

Ablation of plasma membrane Ca2 +-ATPase isoform 4 prevents development of hypertrophy in a model of hypertrophic cardiomyopathy

The mechanisms linking the expression of sarcomeric mutant proteins to the development of pathological hypertrophy in hypertrophic cardiomyopathy (HCM) remain poorly understood. We investigated the role of the plasma membrane Ca2+-ATPase PMCA4 in the HCM phenotype using a transgenic model that expresses mutant (Glu180Gly) α-tropomyosin (Tm180) in heart. Immunoblot analysis revealed that cardiac PMCA4 expression was upregulated early in Tm180 disease pathogenesis. This was accompanied by an increase in levels of the L-type Ca2+-channel, which is implicated in pathological hypertrophy. When Tm180 mice were crossed with a PMCA4-null line, loss of PMCA4 caused the abrogation of hypertrophy in Tm180/PMCA4-null double mutant mice. RT-PCR analysis of Tm180/PMCA4-null hearts revealed blunting of the fetal program and reversion of pro-fibrotic Col1a1 and Col3a1 gene expression to wild-type levels. This was accompanied by evidence of reduced L-type Ca2+-channel expression, and diminished calcineurin activity. Expression of the metabolic substrate transporters glucose transporter 4 and carnitine palmitoyltransferase 1b was preserved and Tm180-related changes in mRNA levels of various contractile stress-related proteins including the cardiac ankyrin protein CARP and the N2B isoform of titin were reversed in Tm180/PMCA4-null hearts. cGMP levels were increased and phosphorylation of vasodilator-stimulated phosphoprotein was elevated in Tm180/PMCA4-null hearts. These changes were associated with a sharp reduction in left ventricular end-diastolic pressure in Tm180/PMCA4-null hearts, which occurred despite persistence of Tm180-related impairment of relaxation dynamics. These results reveal a novel and specific role for PMCA4 in the Tm180 hypertrophic phenotype, with the “protective” effects of PMCA4 deficiency encompassing multiple determinants of HCM-related hypertrophy.

Abstract 279: A Deletion In The N2A Region Of Titin Carried By Muscular Dystrophy With Myositis (mdm) Mice Severely Affects Skeletal Muscle, But Not The Heart

20% of dilated cardiomyopathy patients carry mutations in the giant protein titin. Mutations are predominant in A band but also occur in I band, a domain that regulates passive tension and myocyte signaling. A recessive mouse mutation in titin I band N2A region (mdm) causes early onset muscular dystrophy with myositis and death. We assessed cardiac morphology, function, and transcriptional profiles (RNAseq) in mdm mice. Young homozygous mdm mice (n&gt;6) have reduced body weight (7gms) vs. heterozygous (20gm) or WT (17gm) littermates, with severe skeletal muscle dystrophy. Four-week old homozygous mdm mice have higher left ventricular (LV): body weight ratios. Echocardiography revealed thinner LV posterior wall and septum (LVPWd and IVSd) and normal LV diameter (LVDd); when normalized for body weight, cardiac dimensions were increased compared to WT or heterozygous mdm mice. Fractional shortening was reduced in homozygous Mdm mice (35%) vs. WT (40-41%, p&lt;0.01); histology showed neither overt pathology nor fibrosis. Titin gels showed lack of difference in cardiac titin isoform pattern, consistent with RNAseq, which showed the mdm titin transcript excluded exons 107 and 108, deleting in frame 48 amino acids. 240 transcripts (0.8%) were differentially expressed (fold change &gt;1.5 and &lt;0.75, p&lt;0.001) in homozygous vs. heterozygous mdm hearts; ANP and BNP were mildly upregulated (2- and 1.2-fold). Altered transcripts participated in extracellular and immune signaling pathways. Among titin binding partners, only calpain-3 that interacts with N2A was changed (0.6-fold), consistent with previous reports in skeletal muscle. As humans have heterozygous mutations, we stressed adult heterozygous mdm and WT mice (2 weeks of angiotensin II infusion): both had comparable hypertrophic responses (increased LVPWd and IVSd). Aged (89 week old) unstressed heterozygous mdm mice had normal cardiac dimensions and function. The N2A region, I-band titin mdm mutation causes minimal cardiac dysfunction in mice, unlike the severe skeletal muscle phenotype. Human I-band mutations are unlikely to cause dilated cardiomyopathy.