Mei Methawasin

Experimentally increasing titin compliance in a novel mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole.

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&Dgr;RRMmice). 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&Dgr;RRMmice with a further reduction in homozygous (−/−) mice at only the intact myocyte level. Fibrosis was present in only −/− Rbm20&Dgr;RRM hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20&Dgr;RRM 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.

Shortening of the Elastic Tandem Immunoglobulin Segment of Titin Leads to Diastolic Dysfunction

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.

Shortening of Titin's Elastic Tandem Ig Segment Leads to Diastolic Dysfunction

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.

The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin’s spring elements

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titins 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 titins 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 titins molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.

Deleting titin’s I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function

Significance Mutations in titin are a major cause of heart failure, yet the functions of large parts of titin are not understood. Here we studied titin’s I-band/A-band junction that has been proposed to be crucial for thick filament length control. We made a mouse in which titin’s IA junction was deleted. Super-resolution microscopy (structured illumination microscopy) revealed that deleting the IA junction increases the strain on titin’s molecular spring elements without altering thick filament length. Single cell biomechanical measurements showed that this increases passive stiffness while functional studies at the whole animal level revealed diastolic dysfunction, exercise intolerance, and modest concentric cardiac hypertrophy—signature features of heart failure with preserved ejection fraction. Our studies support that titin is a promising therapeutic target for treating heart failure. 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.

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction

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 &agr;MHC promoter (referred to as cRbm20&Dgr;RRM 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&Dgr;RRM-raloxifene), with dimethyl sulfoxide (DMSO)–injected mice (cRbm20&Dgr;RRM-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&Dgr;RRM-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&Dgr;RRM-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&Dgr;RRM-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&Dgr;RRM-DMSO and cRbm20&Dgr;RRM-raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCAcRbm20&Dgr;RRM-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.

Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titins spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.

Alternative Splicing of Titin Restores Diastolic Function in an HFpEF-Like Genetic Murine Model (TtnΔIAjxn).

RATIONALE Patients with heart failure with preserved ejection fraction (HFpEF) experience elevated filling pressures and reduced ventricular compliance. The splicing factor RNA-binding motif 20 (RBM20) regulates the contour length of titins spring region and thereby determines the passive stiffness of cardiomyocytes. Inhibition of RBM20 leads to super compliant titin isoforms (N2BAsc) that reduce passive stiffness. OBJECTIVE To determine the therapeutic potential of upregulating compliant titin isoforms in an HFpEF-like state in the mouse. METHODS AND RESULTS Constitutive and inducible cardiomyocyte-specific RBM20-inhibited mice were produced on a Ttn(ΔIAjxn) background to assess the effect of upregulating compliant titin at the cellular and organ levels. Genetic deletion of the I-band-A-band junction (IAjxn) in titin increases strain on the spring region and causes a HFpEF-like syndrome in the mouse without pharmacological or surgical intervention. The increased strain represents a mechanical analog of deranged post-translational modification of titin that results in increased passive myocardial stiffness in patients with HFpEF. On inhibition of RBM20 in Ttn(ΔIAjxn) mice, compliant titin isoforms were expressed, diastolic function was normalized, exercise performance was improved, and pathological hypertrophy was attenuated. CONCLUSIONS We report for the first time a benefit from upregulating compliant titin isoforms in a murine model with HFpEF-like symptoms. Constitutive and inducible RBM20 inhibition improves diastolic function resulting in greater tolerance to exercise. No effective therapies exists for treating this pervasive syndrome; therefore, our data on RBM20 inhibition are clinically significant.

The giant protein titin regulates the length of the striated muscle thick filament

The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin’s C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.Thick filaments in skeletal muscle and heart are composed of myosin. The authors show that the length of thick filaments is defined by titin, and that alterations in titin length affect force generation and lead to dilated cardiomyopathy in mice.

Experimentally Increasing Titin Compliance in a Novel Mouse Model Attenuates the Frank-Starling Mechanism but has a Beneficial Effect on Diastole

Background. Experimentally upregulating compliant titins has been suggested as a possible therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study.Here we investigate the effect of upregulating compliant titins on diastolic and systolic function in a novel mouse model that we made with a genetically altered titin splicing factor. We employed integrative approaches from intact cardiomyocytes studied with a carbon-fiber force-measuring system to the left ventricular (LV) chamber studied with pressure(P)-volume(V) analysis and Doppler echocardiography.Methods and Results. Compliant titins were upregulated through deletion of the RNA Recognition Motif of the splicing factor RBM20(Rbm20ΔRRMmice). At both cardiomyocyte and LV chamber levels diastolic stiffness was reduced in heterozygous (+/-) Rbm20ΔRRMmice with a further reduction in homozygous (-/-) mice at only the intact myocyte level. Fibrosis was present in only -/- Rbm20ΔRRM hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20ΔRRM mice, at both the cardiomyocyte and LV chamber levels.Exercise tests revealed an increase in exercise capacity in +/- mice.Conclusions. Results support that titin is not only important in diastolic but also in systolic cardiac function. Upregulating compliant titin isoforms reduces diastolic chamber stiffness due to increased compliance of myocytes but depresses systolic cardiac reserve; under conditions of exercise-induced cardiac stress the beneficial effects on diastolic function dominate. Therapeutic manipulation of the RBM20-based splicing system and careful dosing might be able to avoid effects on fibrosis and systolic function while improving diastolic function of heart failure patients.