Titin Isoform Research Articles

Titin M-line insertion sequence 7 is required for proper cardiac function in mice.

Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation-contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation-contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.

The ryanodine receptor stabilizer S107 ameliorates contractility of adult Rbm20 knockout rat cardiomyocytes.

RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3‐, 6‐, 9‐, and 12‐months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6‐month‐old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy.

Contributions of Myocardial Hypertrophy and Stiffening to Right‐Ventricular Remodeling in a Rat Model of Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive vasculopathy which increases pulmonary vascular stiffness and induces a pressure overload on the right ventricle (RV). In patients with PAH, the RV remodels in response to increased pulmonary pressure, including myocardial hypertrophy and RV geometric remodeling, but can result in intractable right heart failure. Given that the underlying mechanisms of RV remodeling are not well understood,1 we carried out invasive RV catherization in the sugen-hypoxia (SuHx) rat model to assess systolic and diastolic chamber function during the progression of PAH. To distinguish the relative contributions of RV geometric remodeling from myocardial material remodeling to changes in RV function, a biomechanics model was fitted to measured RV pressure-volume relations and morphology after three, five, and eight weeks of sugen injections. After an initial decrease in RV ejection fraction (66±10% to 44±6%, p<0.01), systolic function stabilized (subsequent 2% change in EF through the end of the study, p>0.05), despite progressive increases to RV end-systolic pressure. The model attributed these changes to RV myocardial hypertrophic wall-thickening, with only minor increases in myocyte force generating capacity at week 5 (Figure 1). After RV systolic function stabilized, end-diastolic pressure increased significantly (p<0.05), with maintained RV diastolic volumes. Unlike end systole, RV end-diastolic volume was maintained due to stiffening of myocardium resting properties (Figure 1, panel 2-3). These passive stiffness findings are in agreement with previously identified increases in diastolic chamber and myocardial stiffening in both PAH animals and patients occurring before the onset of severe systolic dysfunction2,3. While significant myocardial passive stiffness was found in this study, there was no evidence of changes in total collagen content (p>0.05) or collagen type I to III ratio (p>0.05). This points towards possible collagen matrix remodeling such as collagen fiber structure, tortuosity, or cross-linking, or changes in titin isoforms or phosphorylation. This progressive increase in RV myocardial passive stiffness that occurs after RV hypertrophy has stabilized systolic function may be a compensatory mechanism to delay or prevent RV dilation but may also eventually contribute to diastolic dysfunction. [1] Van de Veerdonk MC et al. Heart Fail Rev. 2016. [2] Vélez-Rendón D et al. J Biomech Eng. 2019. [3] Rain S et al. Circ Heart Fail. 2016.

Unusual Mobility of the Elastic Protein Titin in Thirteen-Lined Ground Squirrel Hearts

Small mammalian hibernators, such as thirteen-lined ground squirrels, undergo rapid, 100-fold reductions in heart rate, leading to a substantial prolongation of the cardiac cycle when entering torpor. Initial studies using high Urea solubilization buffers and Vertical Agarose Electrophoresis suggest no seasonally dependent change in the ratio of the longer, compliant N2BA to the shorter, stiff N2B cardiac titin isoform (N2BA:N2B). However, an unusually large protein was observed. Previously, this band was observed in preparations using urea-free solubilization buffers and agarose-acrylamide gel systems.

Ribosome profiling analysis of human skeletal muscle identifies reduced translation of mitochondrial proteins with age

ABSTRACT With advancing age, human muscle loses strength and function, but the molecular causes of these losses are unknown. Skeletal muscle shows an age-dependent decline in the levels of different proteins, but whether such decline is associated with reduced translation has not been studied. To address this gap of knowledge, we used the technique of ribosome profiling to study translation in muscle from middle-aged and old individuals. Using ribosome occupancy as a measure of translation status, several mRNAs showed differential translation with age. Older age was associated with lower translation of myosin and titin isoforms and more broadly with the translation of proteins involved in oxidative phosphorylation encoded by the mitochondrial genome. Based on our findings, we propose that mitochondrial proteins are less translated in old skeletal muscle.

Growth hormone-releasing hormone agonists ameliorate chronic kidney disease-induced heart failure with preserved ejection fraction

Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca2+] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome.

Linagliptin prevents left ventricular stiffening by reducing titin cleavage and hypophosphorylation.

The metabolic syndrome (MetS) is an escalating problem worldwide, causing left ventricular stiffening, an early characteristic of diastolic dysfunction for which no treatment exists. As diastolic dysfunction and stiffening in MetS patients are associated with increased circulating dipeptidyl peptidase‐4 (DPP‐4) levels, we investigated whether the clinically approved DPP‐4 inhibitor linagliptin reduces left ventricular stiffness in MetS‐induced cardiac disease. Sixteen‐week‐old obese ZSF1 rats, displaying the MetS and left ventricular stiffness, received linagliptin‐supplemented or placebo diet for four weeks. Linagliptin significantly reduced obesity, hyperlipidaemia, and hyperglycaemia and improved left ventricular relaxation. This improved relaxation was related to decreased cardiac fibrosis and cardiomyocyte passive stiffness (F passive). The reduced F passive was the result of titin isoform switching from the stiff N2B to the more flexible N2BA and increased phosphorylation of total titin and specifically its N2Bus region (S4080 and S3391). Importantly, DPP‐4 directly cleaved titin in vitro, resulting in an increased F passive, which was prevented by simultaneous administration of linagliptin. In conclusion, linagliptin improves left ventricular stiffness in obese ZSF1 rats by preventing direct DPP4‐mediated titin cleavage, as well as by modulating both titin isoform levels and phosphorylation. Reducing left ventricular stiffness by administering linagliptin might prevent MetS‐induced early diastolic dysfunction in human.

C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B–mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B–KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B–KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B–deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

Evaluation of cardiac titin expression after long-term exercise in a rat model

Abstract Introduction The main role of the giant elastic protein titin is to provide passive stiffness to striated (i.e. cardiac and skeletal) muscles. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B isoforms. Titin reduces passive stiffness in cardiac muscle by increased expression of the more compliant N2BA isoform (elevated N2BA:N2B ratio). Moreover, decreased passive stiffness is associated with increased exercise tolerance. Long-term exercise induces physiological adaptation of the heart, termed athlete's heart. Currently, there is limited data of titin's role in the athlete's heart. Aims Our aim is to evaluate exercise-induced morphological and functional changes of the heart. Furthermore, to determine the N2BA:N2B ratio in the rat model of athlete's heart. Methods Rats were divided into exercised (n=12) and control (n=12) groups. Athlete's heart was induced by a 12-week-long swim training (200min/day). The control group swam 5 min/day. Following the training period cardiac changes were assessed by echocardiography. In vivo cardiac function was examined by left ventricular (LV) pressure-volume (P-V) analysis. Titin isoform expressions were detected by sodium-dodecyl-sulfate (SDS)-agarose gel electrophoresis. Results Echocardiography and post-mortem measured cardiomyocyte diameters confirmed athlete's heart and LV hypertrophy in exercised rats. P-V analysis showed improved contractility, active relaxation and mechanoenergetics in the exercised group. The N2BA:N2B titin ratio was significantly increased in exercised rats compared to controls (0.28 vs. 0.21, p&amp;lt;0.05). Conclusions Our results confirm the morphological and functional changes of the athlete's heart. The increased ratio of N2BA:N2B titin corresponds to a more compliant heart in the exercised rats. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Research, Development and Innovation Office of Hungary (K 120277) and the ÚNKP-19-3-I New National Excellence Program of The Ministry For Innovation and Technology.

Differences in stretch-shortening cycle and residual force enhancement between muscles

It has been suggested that cross bridge kinetics and residual force enhancement (RFE) affect force in the stretch-shortening cycle (SSC). Because cross bridge kinetics and titin isoforms, which are thought to be related to RFE, differ between muscles, the SSC effect may be also muscle-dependent. Thus, we compared the SSC effect between psoas and soleus muscles, which have a distinct fiber type distribution and different titin isoforms. Four tests (SSC, SSC control, RFE, RFE control) were conducted using isolated, skinned fibers of psoas and soleus. In the SSC tests, fibers were activated at an average sarcomere length of 2.4 μm, stretched to 3.0 μm, and shortened to 2.4 μm. In the SSC control tests, fibers were activated at an average sarcomere length of 3.0 μm and then shortened to 2.4 μm. The relative increase in mechanical work obtained during shortening between tests was defined as the SSC effect. In the RFE tests, fibers were activated at an average sarcomere length of 2.4 μm and then stretched to 3.0 μm, while the RFE control tests consisted of an isometric contraction at 3.0 μm. The difference in steady-state isometric force between tests was defined as RFE. The SSC effect was greater in soleus than in psoas, while the RFE was the same for both muscles. Since the SSC effect was greater in soleus, while the RFE was the same, the observed greater SSC effect is probably not directly caused by RFE, but may be related to differences in cross bridge kinetics.

Change the Laminin, Change the Cardiomyocyte: Improve Untreatable Heart Failure

No effective medical treatment exists for heart failure with preserved ejection fraction (HFpEF), accounting for approximately half of all heart failure cases. The elevated passive myocardial stiffness in HFpEF is attributed to a combination of alterations in the extracellular matrix (ECM) collagen content and modifications in the sarcomeric protein titin. Here, we propose polylaminin, a biomimetic polymer of laminin, as a promising approach for manipulating the titin isoform shift and phosphorylation in cardiomyocytes. Exploring the pleiotropic effects of polylaminin may be a novel strategy for alleviating symptoms in HFpEF’s multifactorial pathophysiology.

Training Induced Changes to Skeletal Muscle Passive Properties Are Evident in Both Single Fibers and Fiber Bundles in the Rat Hindlimb

Introduction: The passive mechanical behavior of skeletal muscle represents both important and generally underappreciated biomechanical properties with little attention paid to their trainability. These experiments were designed to gain insight into the trainability of muscle passive mechanical properties in both single fibers and fiber bundles. Methods: Rats were trained in two groups: 4 weeks of either uphill (UH) or downhill (DH) treadmill running; with a third group as sedentary control. After sacrifice, the soleus (SOL), extensor digitorum longus (EDL), and vastus intermedius (VI) were harvested. One hundred seventy-nine bundles and 185 fibers were tested and analyzed using a cumulative stretch-relaxation protocol to determine the passive stress and elastic modulus. Titin isoform expression was analyzed using sodium dodecyl sulfate vertical agarose gel electrophoresis (SDS-VAGE). Results: Single fibers: passive modulus and stress were greater for the EDL at sarcomere lengths (SLs) ≥ 3.7 μm (modulus) and 4.0 μm (stress) with DH training compared to UH training and lesser for the SOL (SLs ≥ 3.3 μm) with DH training compared with control; there was no effect of UH training. Vastus intermedius was not affected by either training protocol. Fiber bundles: passive modulus and stress were greater for the EDL at SLs ≥ 2.5 μm (modulus) and 3.3 μm (stress) in the DH training group as compared with control, while no affects were observed in either the SOL or VI for either training group. No effects on titin isoform size were detected with training. Conclusion: This study demonstrated that a trainability of passive muscle properties at both the single fiber and fiber bundle levels was not accompanied by any detectable changes to titin isoform size.

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice.

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

Deleting Titin's C-Terminal PEVK Exons Increases Passive Stiffness, Alters Splicing, and Induces Cross-Sectional and Longitudinal Hypertrophy in Skeletal Muscle.

The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219–225 deleted, the TtnΔ219–225 model (MGI: TtnTM 2.1Mgot). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219–225 muscles: a larger isoform that represents the TtnΔ219–225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219–225 soleus muscle (WT 240 ± 9; TtnΔ219–225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219–225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219–225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin’s PEVK exons 219–225 have high passive tension, due in part to alterations elsewhere in splicing of titin’s spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin’s C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.

Heterozygous loss of Rbm24 in the adult mouse heart increases sarcomere slack length but does not affect function

RNA-binding proteins are key regulators of post-transcriptional processes such as alternative splicing and mRNA stabilization. Rbm24 acts as a regulator of alternative splicing in heart and skeletal muscle, and is essential for sarcomere assembly. Homozygous inactivation of Rbm24 in mice disrupts cardiac development and results in embryonic lethality around E12.5. In the present study, we generated somatic Rbm24 knockout (KO) mice and investigated the effects of reduced levels of Rbm24 in the adult heart. Due to the embryonic lethality of Rbm24 KO mice, we examined cardiac structure and function in adult Rbm24 heterozygotes (HETs). Rbm24 protein expression was 40% downregulated in HET hearts compared to WT hearts. Force measurements on isolated membrane-permeabilized myocytes showed increased sarcomere slack length and lower myofilament passive stiffness in adult Rbm24 HET compared to wildtype cardiomyocytes. As a result of the differences in sarcomere slack length, the relations between force development and sarcomere length differed between WT and Rbm24 HET hearts. No differences in sarcomere structure and titin isoform composition were observed. Likewise, in vivo cardiac function and myocardial structure was unaltered in Rbm24 HET mice compared to WT, at baseline and upon pressure overload after transverse aortic constriction. In conclusion, we generated a somatic Rbm24 KO model and recapitulated the previously reported embryonic phenotype. In adult Rbm24 HET cardiomyocytes we observed increased sarcomere slack length, but no difference in sarcomere structure and cardiac function.

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

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

CaMKIIδ differentially alters the passive mechanics of myocardium

Abnormal diastolic function as seen in failing hearts with preserved ejection fraction (HFpEF) has been linked to alterations in the passive material property of myocardium. Specifically, the non‐crossbridge forming protein named titin contributes a stiffness that varies with fluctuating calcium concentration and has been thought to dominate the passive stiffness of myocardium throughout normal physiological range.Titin is expressed in two isoforms in the heart: the N2B, which has a single spring‐like element, and a more compliant isoform, N2BA, which contains two spring‐like domains (the N2B and N2A). The N2B element exists in all isoforms and is cardiac specific and consists of a large 575 residue unique sequence (N2B‐Us), three flanking immunoglobulin domains, and two other smaller unique sequences. Titin‐based stiffness of myocardium has been shown to be modulated by altered ratio expression of titin isoforms as well as post‐translational modification with unbalanced phosphorylation of cardiac titin characterizing HFpEF. Several proteins can alter titin stiffness including protein kinase (PK) G, extracellular signal‐regulated kinase 2 (ERK‐2), PKC, PKA and calcium calmodulin‐dependent protein kinase II (CaMKII) with varied effects depending on the target site location on titin.CaMKII is a dodecameric enzyme consisting of subunits encoded by four different genes known as CaMKII α,β,γ, and δ. CaMKIIδ is the predominant isoform in the heart and is alternatively spliced in the heart to generate δb and δc differing only by an 11‐amino acid nuclear localization signal. CaMKIIδ heteromultimerization is permissive, however, in that the enzyme may contain subunits from multiple CaMK genes and splice variants of those genes. Thus, it is hypothesized that the general ratio of CaMKIIδ subtypes determine its exact localization within the cardiomyocyte.CaMKIIδ knock‐out has recently been shown to ameliorate heart failure development in response to pressure overload, Gαq expression, and isoproterenol infusion while CaMKIIδ subtypes show a differential regulation of calcium handling in‐vitro and infarct formation following ex‐vivo myocardial ischemia/reperfusion. The idea that the specific CaMKIIδ subtypes differentially regulate cardiac mechanics is interesting yet remains unresolved.In our work, 5–6 weeks old mice models overexpressing CaMKIIδb and CaMKIIδc were bred against a CaMKIIδ knock‐out background, called KOB and KOC respectively. Results indicate that KOB mice show increased passive stiffness (12 mN/mm^2 +− 3 mN/mm^2) compared to KOC passive stiffness (6 mN/mm^2 +− 1 mN/mm^2) at 5–6 weeks prior to heart failure phenotype. Furthermore, as the mice progress into heart failure, the heart passive mechanics of KOB and KOC are consistent, thus, highlighting CaMKII as a precursor to heart failure development.Support or Funding InformationPresident's Disseration Year FellowshipFigure 1

Compliant Titin Isoform Content Is Reduced in Left Ventricles of Sedentary Versus Active Rats.

A sedentary lifestyle is associated with increased cardiovascular risk factors and reduced cardiac compliance when compared to a lifestyle that includes exercise training. Exercise training increases cardiac compliance in humans, but the mechanisms underlying this improvement are unknown. A major determinant of cardiac compliance is the compliance of the giant elastic protein titin. Experimentally reducing titin compliance in animal models reduces exercise tolerance, but it is not known whether sedentary versus chronic exercise conditions cause differences in titin isoform content. We hypothesized that sedentary conditions would be associated with a reduction in the content of the longer, more compliant N2BA isoform relative to the stiffer N2B isoform (yielding a reduced N2BA:N2B ratio) compared to age-matched exercising controls. We obtained left ventricles from 16-week old rats housed for 12 weeks in standard (sedentary) or voluntary running wheel (exercised) housing. The N2BA:N2B ratio was decreased in the hearts of sedentary versus active rats (p = 0.041). Gene expression of a titin mRNA splicing factor, RNA Binding Motif 20 protein (RBM20), correlated negatively with N2BA:N2B ratios (p = 0.006, r = −0.449), but was not different between groups, suggesting that RBM20 may be regulated post-transcriptionally. Total phosphorylation of cardiac titin was not different between the active and sedentary groups. This study is the first to demonstrate that sedentary rats exhibit reduced cardiac titin N2BA:N2B isoform ratios, which implies reduced cardiac compliance. These data suggest that a lack of exercise (running wheel) reduces cardiac compliance and that exercise itself increases cardiac compliance.

The Giant Protein Titin's Role in Cardiomyopathy: Genetic, Transcriptional, and Post-translational Modifications of TTN and Their Contribution to Cardiac Disease.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, sudden cardiac death and heart transplant. DCM is inherited in approximately 50% of cases, in which the most frequent genetic defects are truncation variants of the titin gene (TTNtv). TTN encodes titin, which is the largest protein in the body and is an essential component of the sarcomere. Titin serves as a biological spring, spanning half of the sarcomere and connecting the Z-disk to the M-line, with scaffold and signaling functions. Truncations of titin are believed to lead to either haploinsufficiency and loss-of-function, or to a “poison peptide” effect. However, other titin mechanisms are postulated to influence cardiac function including post-translational modifications, in particular changes in titin phosphorylation that alters the stiffness of the protein, and diversity of alternative splicing that generates different titin isoforms. In this article, we review the role of TTN mutations in development of DCM, how differential expression of titin isoforms relate to DCM pathophysiology, and discuss how post-translational modifications of titin can affect cardiomyocyte function. Current research efforts aim to elucidate the contribution of titin to myofibril assembly, stability, and signal transduction, and how mutant titin leads to cardiac dysfunction and human disease. Future research will need to translate this knowledge toward novel therapeutic approaches that can modulate titin transcriptional and post-translational defects to treat DCM and heart failure.HIGHLIGHTS- Titin (TTN) truncation variants are the most frequent cause of dilated cardiomyopathy, one of the main causes of heart failure and heart transplant. Titin is a giant protein, and the mechanisms causing the disease are both complex and still incompletely understood.- This review discusses the role of titin in myocardial function and in disease. In particular, we discuss TTN gene structure, the complexity of genotype-phenotype correlation in human disease, the physiology of TTN and the role of post-translation modification.- Additional studies will be required to clarify whether missense variants are associated with cardiac disease. While initial studies suggested a role of non-synonymous variants in arrhythmogenic cardiomyopathy, confirmatory investigations have been hampered by the complexity of the protein structure and function.

Effects of Adiponectin on Diastolic Function in Mice Underwent Transverse Aorta Constriction

Diastolic dysfunction is common in various cardiovascular diseases, which could be affected by adiponectin (APN). Nevertheless, the effects of APN on diastolic dysfunction in pressure overload model induced by transverse aorta constriction (TAC) remain to be further elucidated. Here, we demonstrated that treatment of APN attenuated diastolic dysfunction and cardiac hypertrophy in TAC mice. Notably, APN also improved active relaxation of adult cardiomyocytes, increased N2BA/N2B ratios of titin isoform, and reduced collagen type I to type III ratio and lysyl oxidase (Lox) expressions in the myocardial tissue. Moreover, APN supplementation suppressed TAC-induced oxidative stress. In vitro, inhibition of AMPK by compound C (Cpc) abrogated the effect of APN on modulation of titin isoform shift and the anti-hypertrophic effect of APN on cardiomyocytes induced by AngII. In summary, our findings indicate that APN could attenuate diastolic dysfunction in TAC mice, which are at least partially mediated by AMPK pathway.