Titin Isoform Research Articles

Aortic stenosis and aortic regurgitation express different titin isoforms: Differences and relationships with functional and geometric characteristics

Background-Titin represents an important biomechanical sensor which determines compliance and diastolic/systolic function of the left ventricle (LV). To assess the different titin-isoform expression and the relationships with functional and geometric patterns, we analyzed titin-isoform expression and cardiomyocytes contractile function in myocardial biopsy samples of patients undergoing aortic valve replacement (AVR) for aortic stenosis (AS) and for aortic regurgitation (AR).Method -Specimens, collected from the LV of 35 with AS and 35 with AR undergoing AVR were analyzed for titin-isoform expression and cardiomyocytes force measurement. Ten donor hearts were analyzed as controls for normal values. Results were implemented with preoperative geometry and function assessed by Doppler echocardiography.Results-Compared to controls, N2BA/N2B titin-isoforms ratio was reduced to 0.24 in AS (p < 0.001) but increased to 0.51 in AR (p < 0.001). N2BA/N2B titin-isoforms ratio was further reduced in 8 patients with severe (restrictive) diastolic dysfunction (0.17 ± 0.03, p < 0.001) but was increased in patients with severe systolic dysfunction (0.58 ± 0.07, p < 0.001). As compared to controls, Fpasive was higher in AS (6.7 ± 0.2 vs 4.4 ± 0.4 kN/m2, p < 0.001) but was lower in AR (3.7 ± 0.2 vs 4.4 ± 0.4 kN/m2, p < 0.001). Total force was comparable. Fpassive was significantly higher in AS patients with severe than with moderate LV diastolic dysfunction (7.1 ± 0.5 vs 6.6. ± 0.6, p = 0.004).Conclusions-titin-isoform expression differs in AS and AR as adaptive response to different pathophysiologic scenarios. Co-expressing isoforms at varying ratios results in modulation of the passive mechanical behavior of the LV at different degree of dysfunction and allows for compensative adjustment of the diastolic/systolic properties of the myocardium.

Chronic Exercise Increases Compliant Titin and Kettin Isoform Content in Cardiac Muscle of Rat and Drosophila Models

Reduced cardiac passive stiffness correlates with increased exercise tolerance in mammals. Titin, the giant elastic protein, decreases passive stiffness by expression of larger titin isoforms (increased N2BA:N2B ratio) or altered post-translational modifications (increased PKA, decreased PKC phosphorylation). Short-term and acute exercise has been previously shown to change post-translational modifications but not the N2BA:N2B. Kettin is an elastic protein in invertebrates that is also differentially spliced. Little is known about kettin isoform splicing in response to exercise. The purpose of our study was to evaluate changes in titin and kettin isoform content following chronic exercise or sedentary conditions. 4 week old male Sprague-Dawley rats were randomly assigned to cages with (exercise, n=18) and without (sedentary, n=17) running wheels. After 12 weeks, hearts were harvested and the left ventricle solubilized and proteins separated using SDS-VAGE. Titin N2BA:N2B ratio increased (p=0.047) in exercising animals with wheels compared to sedentary animals. Degraded titin to total titin ratio and total titin to myosin heavy chain content ratios were unchanged. Canton S flies (Drosophila) were exercised up to 3 hrs per day 5 times per week for 3 weeks using the PowerTower, a machine that repetitively induces negative geotaxis (climbing exercise). Abdominal segments from whole, flash frozen Drosophila were solubilized from 3 technical replicates containing 10 Drosophila each for both the exercised and un-exercised groups. Abdominal samples contain both cardiac and skeletal muscles, but are more enriched for cardiac muscle than thoraces. Three isoforms of kettin were consistently identified using western blot. Preliminary data showed an increased expression of larger kettin isoforms sizes in exercise versus un-exercised samples. These preliminary studies indicate that both mammalian and invertebrate cardiac tissues have increased compliant isoforms of titin and kettin, respectively.

Pre-Activation of Cardiomyocytes Determines Contractile Force and Speed of Contraction; Role of Titin and Calcium

Cardiomyocytes have, through splicing, an exquisite control over the length of titin, enabling it to regulate passive stiffness. As titin sets the preload of the cardiomyocyte, we hypothesized that together with diastolic Ca2+ it pre-activates the cardiomyocyte during diastole and that this is a major determinant of force production in the subsequent systolic phase. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work-loops that mimic the cardiac cycle in wildtype (WT) and heterozygous (HET) RBM20 deficient rats, with long compliant titin. In addition we studied membrane-permeabilized cardiomyocytes with different amounts of diastolic calcium. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies. HET myocytes operated at higher SL to achieve the same level of work (2.10 µm vs. 1.94 µm at 6 Hz). To delineate the effects of diastolic Ca2+ and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes isolated from rat hearts. In these cells cardiac twitches were simulated by transiently (1 s) exposing the cell to a Ca2+-concentration of 2 µM. Increasing pre-activation by pre-stretching the myocyte increased the kinetics of force development and total force development. Increasing diastolic Ca2+ did increase force development, but only within a small concentration range. These findings are consistent with our hypothesis that pre-activation can increase force development. Highly compliant titin allows cells to function at higher diastolic Ca2+, but this is expected to result in limited contractile reserve.

The Skeletal Muscle Molecular Clock Regulates Titin Splicing and Protein Expression

The circadian factor BMAL1 is a fundamental transcriptional regulator of cell time keeping and expression of cell specific genes important for homeostasis. Our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse to determine the role of this protein in adult skeletal muscle. Skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. Since this change could be caused by changes in the elastic properties of the muscle, we investigated whether titin isoform expression changes in these mice. The tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant shift in expression from a short to long isoform of titin protein (38% long in iMSBmal1-/- vs. 19% in iMSBmal1+/+). RNA-Seq from these muscles indicates increased inclusion of titin exons 150-155 and 190-225, which are found within titin's PEVK region. Because this region is highly extensible and acts as a spring, we performed immunohistochemistry to test whether changes to this protein could have an effect on sarcomere length. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length increased following Bmal1 knockout. To test if increased variability of titin isoform expression similar to the iMSBmal1-/- mouse could lead to variable sarcomere lengths, we are currently transfecting C2C12 myoblasts with U7 snRNPs targeted to titin PEVK regions. Sarcomere length variability will be measured in these cells following 7 days of differentation. Current studies with this model are ongoing and will lead to increased knowledge of titin's role in regulating sarcomere length in a skeletal muscle at rest.

Cardiovascular Function of Modern Pigs Does not Comply with Allometric Scaling Laws

Growing concerns have been expressed regarding cardiovascular performance in modern farm pigs, which has been proposed as a critical factor contributing to the reduced adaptability of modern pigs to stress. Here we tested the hypothesis that cardiac dimensions and pump function in modern heavy farm pigs are disproportionally low for their body weight, and investigated potential underlying mechanisms. The results from the present study indeed demonstrate disproportionally low values for stroke volume and cardiac output in pigs with bodyweights over 150 kg. Importantly, these low values were not the result of impaired left ventricular (LV) systolic contractile function, but were due to a disproportionally small LV end-diastolic volume. The latter was associated with changes in determinants of LV passive stiffness, including (i) an increase in LV myocardial collagen, (ii) a shift from the compliant N2BA titin isoform towards the stiff N2B, and (iii) a marked elevation of aortic blood pressure. Taken together, these results demonstrate reduced pumping capacity of the hearts of heavy modern pigs, due to structural abnormalities in the LV myocardium.

Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy.

Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM) is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca2+-sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.

Placenta-Derived Adherent Stromal Cells Improve Diabetes Mellitus-Associated Left Ventricular Diastolic Performance.

Left ventricular (LV) diastolic dysfunction is among others attributed to cardiomyocyte stiffness. Mesenchymal stromal cells (MSC) have cardiac‐protective properties. We explored whether intravenous (i.v.) application of PLacenta‐eXpanded (PLX) MSC‐like cells (PLX) improves LV diastolic relaxation in streptozotocin (STZ)‐induced diabetic mice and investigated underlying mechanisms. Diabetes mellitus was induced by STZ application (50 mg/kg body weight) during five subsequent days. One week after the first STZ injection, PLX or saline were i.v. applied. Two weeks later, mice were hemodynamically characterized and sacrificed. At this early stage of diabetic cardiomyopathy with low‐grade inflammation and no cardiac fibrosis, PLX reduced LV vascular cell adhesion molecule‐1, transforming growth factor‐β1, and interferon‐γ mRNA expression, induced the percentage of circulating regulatory T cells, and decreased the splenic pro‐fibrotic potential in STZ mice. STZ + PLX mice exhibited higher LV vascular endothelial growth factor mRNA expression and arteriole density versus STZ mice. In vitro, hyperglycemic PLX conditioned medium restored the hyperglycemia‐impaired tube formation and adhesion capacity of human umbelical vein endothelial cells (HUVEC) via increasing nitric oxide (NO) bioavailability. PLX further induced the diabetes‐downregulated activity of the NO downstream protein kinase G, as well as of protein kinase A, in STZ mice, which was associated with a raise in phosphorylation of the titin isoforms N2BA and N2B. Concomitantly, the passive force was lower in single isolated cardiomyocytes from STZ + PLX versus from STZ mice, which led to an improvement of LV diastolic relaxation. We conclude that i.v. PLX injection improves diabetes mellitus‐associated diastolic performance via decreasing cardiomyocyte stiffness. Stem Cells Translational Medicine 2017;6:2135–2145

Titin isoforms are increasingly protected against oxidative modifications in developing rat cardiomyocytes

During the perinatal adaptation process N2BA titin isoforms are switched for N2B titin isoforms leading to an increase in cardiomyocyte passive tension (Fpassive). Here we attempted to reveal how titin isoform composition and oxidative insults (i.e. sulfhydryl (SH)-group oxidation or carbonylation) influence Fpassive of left ventricular (LV) cardiomyocytes during rat heart development. Moreover, we also examined a hypothetical protective role for titin associated small heat shock proteins (sHSPs), Hsp27 and αB-crystallin in the above processes. Single, permeabilized LV cardiomyocytes of the rat (at various ages following birth) were exposed either to 2,2′-dithiodipyridine (DTDP) to provoke SH-oxidation or Fenton reaction reagents (iron(II), hydrogen peroxide (H2O2), ascorbic acid) to induce protein carbonylation of cardiomyocytes in vitro. Thereafter, cardiomyocyte force measurements for Fpassive determinations and Western immunoblot assays were carried out for the semiquantitative determination of oxidized SH-groups or carbonyl-groups of titin isoforms and to monitor sHSPs’ expressions. DTDP or Fenton reagents increased Fpassive in 0- and 7-day-old rats to relatively higher extents than in 21-day-old and adult animals. The degrees of SH-group oxidation or carbonylation declined with cardiomyocyte age to similar extents for both titin isoforms. Moreover, the above characteristics were mirrored by increasing levels of HSP27 and αB-crystallin expressions during cardiomyocyte development. Our data implicate a gradual build-up of a protective mechanism against titin oxidation through the upregulation of HSP27 and αB-crystallin expressions during postnatal cardiomyocyte development.

Myofilament Remodeling and Function Is More Impaired in Peripartum Cardiomyopathy Compared with Dilated Cardiomyopathy and Ischemic Heart Disease

Peripartum cardiomyopathy (PPCM) and dilated cardiomyopathy (DCM) show similarities in clinical presentation. However, although DCM patients do not recover and slowly deteriorate further, PPCM patients show either a fast cardiac deterioration or complete recovery. The aim of this study was to assess if underlying cellular changes can explain the clinical similarities and differences in the two diseases. We, therefore, assessed sarcomeric protein expression, modification, titin isoform shift, and contractile behavior of cardiomyocytes in heart tissue of PPCM and DCM patients and compared these with nonfailing controls. Heart samples from ischemic heart disease (ISHD) patients served as heart failure control samples. Passive force was only increased in PPCM samples compared with controls, whereas PPCM, DCM, and ISHD samples all showed increased myofilament Ca2+ sensitivity. Length-dependent activation was significantly impaired in PPCM compared with controls, no impairment was observed in ISHD samples, and DCM samples showed an intermediate response. Contractile impairments were caused by impaired protein kinase A (PKA)-mediated phosphorylation because exogenous PKA restored all parameters to control levels. Although DCM samples showed reexpression of EH-myomesin, an isoform usually only expressed in the heart before birth, PPCM and ISHD did not. The lack of EH-myomesin, combined with low PKA-mediated phosphorylation of myofilament proteins and increased compliant titin isoform, may explain the increase in passive force and blunted length-dependent activation of myofilaments in PPCM samples.

P.475 - Alternative splicing in titin: new insights into exon usage

TTN gene contains 364 exons and encodes titin, a giant protein with multiple functions in skeletal and cardiac muscles. As a result of alternative splicing, several titin isoforms are expressed. TTN isoforms can be divided into three main classes based on the presence of the N2A and N2B elements in the I-band region. N2A isoforms, characterized by the inclusion of the N2A element and exclusion of the cardiac-specific N2B element, are expressed in the skeletal muscles. N2B (N2B element only) and N2BA (including both the N2B and N2A elements) isoforms are expressed in the heart. Further variability is seen within each class as a consequence of a variable exon usage in different muscles and across developmental and physiological states. With over 1 million potential splicing variants, TTN could be the most alternatively spliced human gene. Expression data obtained from microarray, RNA-seq and RT-PCRs reveals new insights into the TTN exon usage. A better understanding of TTN alternative transcripts, their biological meaning and their specific tissue expression is crucial for the clinical interpretation of molecular findings.

The link between exercise and titin passive stiffness

What is the topic of this review? This review focuses on how in vivo and molecular measurements of cardiac passive stiffness can predict exercise tolerance and how exercise training can reduce cardiac passive stiffness. What advances does it highlight? This review highlights advances in understanding the relationship between molecular (titin-based) and in vivo (left ventricular) passive stiffness, how passive stiffness modifies exercise tolerance, and how exercise training may be therapeutic for cardiac diseases with increased passive stiffness. Exercise can help alleviate the negative effects of cardiovascular disease and cardiovascular co-morbidities associated with sedentary behaviour; this may be especially true in diseases that are associated with increased left ventricular passive stiffness. In this review, we discuss the inverse relationship between exercise tolerance and cardiac passive stiffness. Passive stiffness is the physical property of cardiac muscle to produce a resistive force when stretched, which, in vivo, is measured using the left ventricular end diastolic pressure-volume relationship or is estimated using echocardiography. The giant elastic protein titin is the major contributor to passive stiffness at physiological muscle (sarcomere) lengths. Passive stiffness can be modified by altering titin isoform size or by post-translational modifications. In both human and animal models, increased left ventricular passive stiffness is associated with reduced exercise tolerance due to impaired diastolic filling, suggesting that increased passive stiffness predicts reduced exercise tolerance. At the same time, exercise training itself may induce both short- and long-term changes in titin-based passive stiffness, suggesting that exercise may be a treatment for diseases associated with increased passive stiffness. Direct modification of passive stiffness to improve exercise tolerance is a potential therapeutic approach. Titin passive stiffness itself may be a treatment target based on the recent discovery of RNA binding motif 20, which modifies titin isoform size and passive stiffness. Translating these discoveries that link exercise and left ventricular passive stiffness may provide new methods to enhance exercise tolerance and treat patients with cardiovascular disease.

Abstract 69: Cardiac Fibrosis Induced by Mkk6-p38 Activation Alters Myocardial Stiffness and Myofilament Function

Heart failure with preserved ejection fraction (HFpEF) accounts for at least half of the patients with heart failure, but to date there are no proven therapies for these patients. Cardiac fibrosis and increased collagen content plays an important role in the pathophysiology of HFpEF. Studies in human HFpEF patients have indicated increased cardiac fibrosis, myocardial stiffness and myofilament calcium sensitivity. However, it is unclear how and if cardiac fibrosis directly alters myofilament function. In this study we used a new model of cardiac fibrosis genetically induced by the constitutive activation of MKK6-p38 (MKK6-Tg) signaling specifically in resident cardiac fibroblasts to determine the effect of fibrosis on myofilament stiffness and function. The MKK6-Tg mice develop interstitial and perivascular fibrosis in the heart accompanied with severe diastolic dysfunction, although systolic function remains normal. We measured the passive stiffness and isometric contractile properties of demembranated papillary muscle preparations. Our results show an increase in passive stiffness in demembranated papillary muscles from MKK6-Tg mice compared to NTG littermates when stretched to 24% from sarcomere length of 2.0 μM. Passive tension at SL=2.3 was significantly increased in MKK6-Tg mice (28±4 vs. 13±3 mN/mm 2 , P&lt;0.05) compared to NTG mice, which was ascribed to changes in Titin isoform as well as extracellular matrix composition. The MKK6-Tg mice also show an increase in Ca 2+ sensitivity (pCa 50 =5.72±0.05 vs. 5.56±0.02, P&lt;0.05). This was associated with an increased rate of force redevelopment in MKK6-Tg mice compared to NTG littermates (k TR =35±8 vs. 22±3 s -1 ). Towards understanding the mechanism of these myofilament changes we performed phosphate affinity electrophoresis and saw decreased phosphorylation of both Troponin I and Troponin T in MKK6-Tg hearts. There was no change in troponin I phosphorylation level at Ser23/24, indicating role of none-PKA mediated phosphorylation in mechanical changes seen in this model.

Abstract 393: Cronos Titin in Human Heart Development

Titin is a giant myofibrillar protein that provides passive tension and facilitates sarcomere formation in striated muscle, where it spans from the Z-disk to the M-line. Recently, an alternative start site has been identified in the titin gene that is predicted to allow for expression of a novel isoform, Cronos, that is ~2/3 the size of full-length titin and lacks the Z-disk and most of the I-band domains. Because the start site for this isoform is spliced out of full-length titin transcripts, we are able to determine the expression levels of Cronos compared to full-length titin. Reverse-transcription followed by quantitative PCR using primers specific to this region indicate Cronos transcript levels are higher in human fetal ventricle and atrial tissue (days 90-105) than in adult samples, while full-length titin transcripts are higher in adult than fetal cardiac tissue. Additionally, human ventricular tissue stained with a custom antibody raised to recognize this specific region of Cronos titin indicated high levels of protein expression and integration into myofibrils in early fetal samples (days 54 and 81), while this novel isoform was nearly undetectable in later fetal (day 130) and adult tissue. The Cronos titin transcript and protein were also detected in high quantities in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which are believed to have a fetal level of maturity. Based on these data, we conclude that Cronos is predominantly a fetal isoform of titin. Future experiments will investigate the effects of a Cronos titin knockdown in hiPSC-CMs during and after differentiation to establish its functional role in human heart development.

TITINdb-a computational tool to assess titin's role as a disease gene.

SummaryLarge numbers of rare and unique titin missense variants have been discovered in both healthy and disease cohorts, thus the correct classification of variants as pathogenic or non-pathogenic has become imperative. Due to titin’s large size (363 coding exons), current web applications are unable to map titin variants to domain structures. Here, we present a web application, TITINdb, which integrates titin structure, variant, sequence and isoform information, along with pre-computed predictions of the impact of non-synonymous single nucleotide variants, to facilitate the correct classification of titin variants.Availability and implementationTITINdb can be freely accessed at http://fraternalilab.kcl.ac.uk/TITINdbSupplementary information Supplementary data are available at Bioinformatics online.

Insulin regulates titin pre-mRNA splicing through the PI3K-Akt-mTOR kinase axis in a RBM20-dependent manner

Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20 (RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-Akt-mTOR kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels, while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.

TTN genotype is associated with fascicle length and marathon running performance.

Titin provides a molecular blueprint for muscle sarcomere assembly, and sarcomere length can vary according to titin isoform expression. If variations in sarcomere length influence muscle fascicle length, this may provide an advantage for running performance. Thus, the aim of this study was to investigate whether the titin (TTN) rs10497520 polymorphism was associated with muscle fascicle length in recreationally active men (RA; n=137) and marathon personal best time in male marathon runners (MR; n=141). Fascicle length of the vastus lateralis was assessed in vivo using B-mode ultrasonography at 50% of muscle length in RA. All participants provided either a whole blood, saliva or buccal cell sample, from which DNA was isolated and genotyped using real-time polymerase chain reaction. Vastus lateralis fascicle length was 10.4% longer in CC homozygotes, those carrying two copies of the C-allele, than CT heterozygotes (P=.003) in RA. In the absence of any TT homozygotes, reflective of the low T-allele frequency within Caucasian populations, it is unclear whether fascicle length for this group would have been smaller still. No differences in genotype frequency between the RA and MR groups were observed (P=.500), although within the MR group, the T-allele carriers demonstrated marathon personal best times 2minutes 25seconds faster than CC homozygotes (P=.020). These results suggest that the T-allele at rs10497520 in the TTN gene is associated with shorter skeletal muscle fascicle length and conveys an advantage for marathon running performance in habitually trained men.

Impact of titin strain on the cardiac slow force response

Stretch of myocardium, such as occurs upon increased filling of the cardiac chamber, induces two distinct responses: an immediate increase in twitch force followed by a slower increase in twitch force that develops over the course of several minutes. The immediate response is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL). The slowly developing force response, termed the Slow Force Response (SFR), is caused by a slowly developing increase in intracellular Ca2+ upon sustained stretch. A blunted immediate force response was recently reported for myocardium isolated from homozygous giant titin mutant rats (HM) compared to muscle from wild-type littermates (WT). Here, we examined the impact of titin isoform on the SFR. Right ventricular trabeculae were isolated and mounted in an experimental chamber. SL was measured by laser diffraction. The SFR was recorded in response to a 0.2 μm SL stretch in the presence of [Ca2+]o = 0.4 mM, a bathing concentration reflecting ∼50% of maximum twitch force development at 25 °C. Presence of the giant titin isoform (HM) was associated with a significant reduction in diastolic passive force upon stretch, and ∼50% reduction of the magnitude of the SFR; the rate of SFR development was unaffected. The sustained SL stretch was identical in both muscle groups. Therefore, our data suggest that cytoskeletal strain may underlie directly the cellular mechanisms that lead to the increased intracellular [Ca2+]i that causes the SFR, possibly by involving cardiac myocyte integrin signaling pathways.

RBM20, a potential target for treatment of cardiomyopathy via titin isoform switching

Cardiomyopathy, also known as heart muscle disease, is an unfavorable condition leading to alterations in myocardial contraction and/or impaired ability of ventricular filling. The onset and development of cardiomyopathy have not currently been well defined. Titin is a giant multifunctional sarcomeric filament protein that provides passive stiffness to cardiomyocytes and has been implicated to play an important role in the origin and development of cardiomyopathy and heart failure. Titin-based passive stiffness can be mainly adjusted by isoform switching and post-translational modifications in the spring regions. Recently, genetic mutations of TTN have been identified that can also contribute to variable passive stiffness, though the detailed mechanisms remain unclear. In this review, we will discuss titin isoform switching as it relates to alternative splicing during development stages and differences between species and muscle types. We provide an update on the regulatory mechanisms of TTN splicing controlled by RBM20 and cover the roles of TTN splicing in adjusting the diastolic stiffness and systolic compliance of the healthy and the failing heart. Finally, this review attempts to provide future directions for RBM20 as a potential target for pharmacological intervention in cardiomyopathy and heart failure.

Genotype-specific pathogenic effects in human dilated cardiomyopathy.

Key points Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy. TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+‐sensitivity and reduced length‐dependent activation. TNNT2p.K217del caused increased passive tension.A mutation in the gene encoding Lamin A/C (LMNA p.R331Q) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy.Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non‐sarcomeric genes. In this study we defined the pathogenic effects of three DCM‐causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation) and cardiac troponin T (TNNT2p.K217deletion; also known as the p.K210del) and the non‐sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane‐permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+‐sensitivity and impaired length‐dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild‐type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.

Novex-3, the tiny titin of muscle.

The giant multi-functional striated muscle protein titin is the third most abundant muscle protein after myosin and actin. Titin plays a pivotal role in myocardial passive stiffness, structural integrity and stress-initiated signaling pathways. The complete sequence of the human titin gene contains three isoform-specific mutually exclusive exons [termed novel exons (novex)] coding for the I-band sequence, named novex-1 (exon 45), novex-2 (exon 46) and novex-3 (exon 48). Transcripts containing either the novex-1 or novex-2 exons code for the novex-1 and novex-2 titin isoforms. The novex-3 transcript contains a stop codon and polyA tail signal, resulting in an unusually small (∼700 kDa) isoform, referred to as novex-3 titin. This 'tiny titin' isoform extends from the Z-disc (N-terminus) to novex-3 (C-terminus) and is expressed in all striated muscles. Biochemical analysis of novex-3 titin in cardiomyocytes shows that obscurin, a vertebrate muscle protein, binds to novex-3 titin. The novex-3/obscurin complex localizes to the Z-disc region and may regulate calcium, and SH3- and GTPase-associated myofibrillar signaling pathways. Therefore, novex-3 titin could be involved in stress-initiated sarcomeric restructuring.