Alternative splicing plays a major role in the adaptation of cardiac function exemplified by the isoform switch of titin, which adjusts ventricular filling. We previously identified a rat strain deficient in titin splicing. Using genetic mapping, we found a loss-of-function mutation in RBM20 as the underlying cause for the pathological titin isoform expression. Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. We showed that the phenotype of Rbm20 deficient rats resembles the human pathology. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20 dependent regulation of alternative splicing. Additionally to titin we identified a set of 30 genes with conserved regulation between human and rat. This network is enriched for genes previously linked to cardiomyopathy, ion-homeostasis, and sarcomere biology. Our studies emphasize the importance of posttranscriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

second model, the two main conditions were parametrically modulated by the two categories, respectively (SOM, S5.1). The activation of the precuneus was higher for hard dominance-solvable games than for easy ones ( Fig. 4A and table S10). The activation of the insula was higher for the highly focal coordination games than for less focal ones ( Fig. 4B and table S11). Previous studies also found that precuneus activity increased when the number of planned moves increased (40, 41). The higher demand for memory-related imagery and memory retrieval may explain the greater precuneus activation in hard dominance-solvable games. In highly focal coordination games, the participants may have felt quite strongly that the pool students must notice the same salient feature. This may explain why insula activation correlates with NCI.Participants might have disagreed about which games were difficult. We built a third model to investigate whether the frontoparietal activation correlates with how hard a dominance-solvable game is and whether the activation in insula and ACC correlates with how easy a coordination game is. Here, the two main conditions were parametrically modulated by each participant's probability of obtaining a reward in each game (SOM, S2.2 and S5.2). We found a negative correlation between the activation of the precuneus and the participant's probability of obtaining a reward in dominance-solvable games ( Fig. 4C and table S12), which suggests that dominance-solvable games that yielded lower payoffs presented harder mental challenges. In a previous study on working memory, precuneus activity positively correlated with response times, a measure of mental effort (24). Both findings are consistent with the interpretation that subjective measures reflecting harder tasks (higher efforts) correlate with activation in precuneus. A positive correlation between insula activation and the participant's probability of obtaining a reward again suggests that coordination games with a highly salient feature strongly activated the "gut feeling" reported by many participants (Fig. 4D and table S13). A previous study found that the subjective rating of "chills intensity" in music correlates with activation of insula (42). Both findings are consistent with the interpretation that the subjective intensity of how salient a stimulus is correlates with activation in insula.As mentioned, choices were made significantly faster in coordination games than in dominancesolvable games. The results of the second and third models provide additional support for the idea that intuitive and deliberative mental processes have quite different properties. The "slow and effortful" process was more heavily taxed when the dominance-solvable games were harder. The "fast and effortless" process was more strongly activated when coordination was easy.

Abstract-Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (ϩ/Ϫ) and homozygous (Ϫ/Ϫ) for the knockout allele. Wild-type (ϩ/ϩ), cMyBP-C ϩ/Ϫ , and cMyBP-C Ϫ/Ϫ mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C ϩ/Ϫ mice were indistinguishable from wild-type littermates, cMyBP-C Ϫ/Ϫ mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C Ϫ/Ϫ mice. Ca 2ϩ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C Ϫ/Ϫ but not cMyBP-C ϩ/Ϫ mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function. Key Words: myosin binding protein-C Ⅲ heart Ⅲ myocardium Ⅲ gene knockout Ⅲ sarcomeric proteins M yosin binding protein-C (MyBP-C), also known as C-protein, 1 is a thick filament accessory protein that is present in nearly all vertebrate striated muscles but whose function is unknown. Nonetheless, there is compelling evidence to suggest that MyBP-C is a significant determinant of muscle contractile properties. In particular, cardiac MyBP-C (cMyBP-C) is a target for phosphorylation in response to various inotropic stimuli, including sympathetic stimuli that effect trisphosphorylation of cMyBP-C via cAMP-dependent protein kinase (PKA). 2 In addition, mutations of the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy (FHC), 3 an inherited disorder linked to mutations in cardiac contractile proteins (for review, see Bonne et al 4 and Seidman and Seidman 5 ).However, despite clues suggesting the importance of cMyBP-C to cardiac health, the function of cMyBP-C has remained enigmatic. For instance, although numerous studies have investigated effects of PKA on cardiac contractility (eg, Strang et al 6 and Patel et al 7 ), the role, if any, of cMyBP-C in mediating contractile responses to PKA has been difficult to discern. 8 -10 Similarly, the mechanisms by which cMyBP-C mutations affect cardiac function are not well understo...

Abstract-Normal cardiac function requires dynamic modulation of contraction. ␤1-Adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k rel (s-1), and delayed force development, k df (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k rel and k df similar to WT myocardium, but, unlike WT, k rel and k df were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction. Key Words: cMyBP-C Ⅲ phosphorylation Ⅲ contraction kinetics T he strength and kinetics of cardiac contraction vary on a beat-to-beat basis as a way to match cardiac output to the circulatory demands of the body. Reduced capacity to modulate contraction has long been recognized as a key feature of dysfunction in heart failure 1 and more recently in hypertrophic cardiomyopathy (HCM). 2 This study explores the possibility that phosphorylation of cardiac myosin binding protein (cMyBP)-C modulates contraction in skinned and living myocardium.MyBP-C is a component of the thick filament in striated muscle 3 and is evident as 7 to 9 bands at 43-nm intervals 4 within the center of each half-thick filament in the A-band. Its location at every third crossbridge crown, ie, every 42.9 nm 5 suggests that cMyBP-C has a regulatory role with respect to thick filament activity. Unlike the skeletal muscle isoform, cMyBP-C is readily phosphorylated by protein kinase (PK)A, 6,7 calcium calmodulin kinase (CaMK) II, 6,7 and PKC. 8,9 Phosphorylation of cMyBP-C may promote actin-myosin interactions by either relieving a structural constraint on myosin to allow closer proximity with actin 10,11 or reducing the binding of cMyBP-C to the S2 domain of myosin to allow greater flexibility...

The Ca 2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83: 3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC~) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca 2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (< 1 ms) released to a shorter length and then reextended by ~200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 -e -~) to obtain the value of kt~. In control fibers, ktr increased sigmoidally with increases in [Ca 2+] ; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 + 1.2 s-1 at 15~ than in rabbit psoas fibers (18.7 _+ 1.0 s-l). Phosphorylation of LC~ was accomplished by repeated Ca 2+ activations (pCa 4.5) of the fibers in solutions containing 6 #M calmodulin and 0.5/~M myosin light chain kinase, a protocol that resulted in an increase in LC~ phosphorylation from ~ 10% in the control fibers to >80% after treatment. After phosphorylation, kt~ was unchanged at maximum or very low levels of Ca 2+ activation. However, at intermediate levels of Ca ~ § activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC~ phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca 2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology.86:585-600), the effect of LC2 phosphorylation to increase the Ca ~+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver. Readdition of purified troponin C to the extracted fibers restored the effect of phosphorylation to potentiate force at submaximal levels of Ca ~+ activation. Thus, the mechanism of phosphorylation-dependent increases in steady-state isometric force appears to involve modulation of the effect of cross-bridges bound to actin to cooperatively enhance the Ca ~+ activation of the thin filament. The observation of a phosphorylationdependent increase in the Ca 2+ sensitivity of k,r likely has important implications in terms of dynamic contractile funct...