Grace M. Arteaga

Dilated Cardiomyopathy Mutant Tropomyosin Mice Develop Cardiac Dysfunction With Significantly Decreased Fractional Shortening and Myofilament Calcium Sensitivity

Mutations in striated muscle &agr;-tropomyosin (&agr;-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within &agr;-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of &agr;-TM mutations associated with DCM, we generated transgenic mice that express mutant &agr;-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous &agr;-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca2+ sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of &bgr;-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca2+ handling proteins sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor. Furthermore, our study also indicates that the &agr;-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca2+ sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.

Attenuation of length dependence of calcium activation in myofilaments of transgenic mouse hearts expressing slow skeletal troponin I.

1 We compared sarcomere length (SL) dependence of the Ca2+‐force relation of detergent‐extracted bundles of fibres dissected from the left ventricle of wild‐type (WT) and transgenic mouse hearts expressing slow skeletal troponin I (ssTnI‐TG). Fibre bundles from the hearts of the ssTnI‐TG demonstrated a complete replacement of the cardiac troponin I (cTnI) by ssTnI. 2 Compared to WT controls, ssTnI‐TG fibre bundles were more sensitive to Ca2+ at both short SL (1.9 ± 0.1 μm) and long SL (2.3 ± 0.1 μm). However, compared to WT controls, the increase in Ca2+ sensitivity (change in half‐maximally activating free Ca2+; ΔEC50) associated with the increase in SL was significantly blunted in the ssTnI‐TG myofilaments. 3 Agents that sensitize the myofilaments to Ca2+ by promoting the actin‐myosin reaction (EMD 57033 and CGP‐48506) significantly reduced the length‐dependent ΔEC50 for Ca2+ activation, when SL in WT myofilaments was increased from 1.9 to 2.3 μm. 4 Exposure of myofilaments to calmidazolium (CDZ), which binds to cTnC and increases its affinity for Ca2+, sensitized force developed by WT myofilaments to Ca2+ at SL 1.9 μm and desensitized the WT myofilaments at SL 2.3 μm. There were no significant effects of CDZ on ssTnI‐TG myofilaments at either SL. 5 Our results indicate that length‐dependent Ca2+ activation is modified by specific changes in thin filament proteins and by agents that promote the actin‐myosin interaction. Thus, these in vitro results provide a basis for using these models to test the relative significance of the length dependence of activation in situ.

Expression of slow skeletal troponin I in adult transgenic mouse heart muscle reduces the force decline observed during acidic conditions

1 Acidosis in cardiac muscle is associated with a decrease in developed force. We hypothesized that slow skeletal troponin I (ssTnI), which is expressed in neonatal hearts, is responsible for the observed decreased response to acidic conditions. To test this hypothesis directly, we used adult transgenic (TG) mice that express ssTnI in the heart. Cardiac TnI (cTnI) was completely replaced by ssTnI either with a FLAG epitope introduced into the N‐terminus (TG‐ssTnI*) or without the epitope (TG‐ssTnI) in these mice. TG mice that express cTnI were also generated as a control TG line (TG‐cTnI). Non‐transgenic (NTG) littermates were used as controls. 2 We measured the force‐calcium relationship in all four groups at pH 7.0 and pH 6.5 in detergent‐extracted fibre bundles prepared from left ventricular papillary muscles. The force‐calcium relationship was identical in fibre bundles from NTG and TG‐cTnI mouse hearts, therefore NTG mice served as controls for TG‐ssTnI* and TG‐ssTnI mice. Compared to NTG controls, the force generated by fibre bundles from TG mice expressing ssTnI was more sensitive to Ca2+. The shift in EC50 (the concentration of Ca2+ at which half‐maximal force is generated) caused by acidic pH was significantly smaller in fibre bundles isolated from TG hearts compared to those from NTG hearts. However, there was no difference in the force‐calcium relationship between hearts from the TG‐ssTnI* and TG‐ssTnI groups. 3 We also isolated papillary muscles from the right ventricle of NTG and TG mouse hearts expressing ssTnI and measured isometric force at extracellular pH 7.33 and pH 6.75. At acidic pH, after an initial decline, twitch force recovered to 60 ± 3 % (n= 7) in NTG papillary muscles, 98 ± 2 % (n= 5) in muscles from TG‐ssTnI* and 96 ± 3 % (n= 7) in muscles from TG‐ssTnI hearts. Our results indicate that TnI isoform composition plays a crucial role in the determination of myocardial force sensitivity to acidosis.

Expression of Slow Skeletal Troponin I in Hearts of Phospholamban Knockout Mice Alters the Relaxant Effect of β-Adrenergic Stimulation

&bgr;-Adrenergic stimulation of the heart results in an enhanced relaxation rate in association with phosphorylation of both cardiac troponin I (cTnI) and phospholamban (PLB). We studied new lines of mice generated by crossbreeding mice that express slow skeletal troponin I (ssTnI) with PLB knockout (PLBKO) mice. This crossbreeding resulted in the generation of PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with isoproterenol (ISO) significantly increased the peak amplitude of fura-2 ratio in PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI groups of mice. However, in the presence of ISO, there were no differences in the peak amplitude of fura-2 ratio among cells isolated from hearts of PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. In cells from PLB/cTnI mice, the extent of shortening was increased and the time of relaxation was significantly decreased during &bgr;-adrenergic stimulation. In PLBKO/cTnI cells, stimulation with ISO resulted in an increased extent of shortening and no change in time of relaxation. However, stimulation with ISO in cells isolated from PLBKO/ssTnI mice not only significantly increased the extent of cell shortening but also increased the time of relaxation. We also determined the kinetics of relaxation of papillary muscles isolated from all four groups of animals in the presence and absence of ISO. Perfusion with ISO increased the rate of relaxation only in PLB/cTnI, PLB/ssTnI, and PLBKO/cTnI muscles. During ISO stimulation, the time of relaxation was unchanged in PLBKO/ssTnI muscles. Our data directly demonstrate that phosphorylation of both PLB and cTnI contributes to increased rate of relaxation during &bgr;-adrenergic stimulation.

Interns' Success With Clinical Procedures in Infants After Simulation Training

BACKGROUND AND OBJECTIVE: Simulation-based medical education (SBME) is used to teach residents. However, few studies have evaluated its clinical impact. The goal of this study was to evaluate the impact of an SBME session on pediatric interns’ clinical procedural success. METHODS: This randomized trial was conducted at 10 academic medical centers. Interns were surveyed on infant lumbar puncture (ILP) and child intravenous line placement (CIV) knowledge and watched audiovisual expert modeling of both procedures. Participants were randomized to SBME mastery learning for ILP or CIV and for 6 succeeding months reported clinical performance for both procedures. ILP success was defined as obtaining a sample on the first attempt with <1000 red blood cells per high-power field or fluid described as clear. CIV success was defined as placement of a functioning catheter on the first try. Each group served as the control group for the procedure for which they did not receive the intervention. RESULTS: Two-hundred interns participated (104 in the ILP group and 96 in the CIV group). Together, they reported 409 procedures. ILP success rates were 34% (31 of 91) for interns who received ILP mastery learning and 34% (25 of 73) for controls (difference: 0.2% [95% confidence interval: –0.1 to 0.1]). The CIV success rate was 54% (62 of 115) for interns who received CIV mastery learning compared with 50% (58 of 115) for controls (difference: 3% [95% confidence interval: –10 to 17]). CONCLUSIONS: Participation in a single SBME mastery learning session was insufficient to affect pediatric interns’ subsequent procedural success.

Rituximab for successful management of probable pediatric catastrophic antiphospholipid syndrome

Catastrophic antiphospholipid syndrome (CAPS) is a life‐threatening condition characterized by small‐vessel thrombi and a rapid onset of multiorgan system failure associated with systemic inflammatory response syndrome. Current treatment options include anticoagulants, corticosteroids, plasma exchange, and intravenous immunoglobulin, but these are not always effective. Rituximab, a chimeric anti‐CD20 monoclonal antibody, may help eliminate autoreactive B cells and thus limit the rapid inflammatory process involved in CAPS. We describe the use of rituximab in the successful initial management of a probable case of pediatric CAPS. Pediatr Blood Cancer 2009;52:536–538.

Impact of Just-in-Time and Just-in-Place Simulation on Intern Success With Infant Lumbar Puncture.

BACKGROUND AND OBJECTIVE: Simulation-based skill trainings are common; however, optimal instructional designs that improve outcomes are not well specified. We explored the impact of just-in-time and just-in-place training (JIPT) on interns’ infant lumbar puncture (LP) success. METHODS: This prospective study enrolled pediatric and emergency medicine interns from 2009 to 2012 at 34 centers. Two distinct instructional design strategies were compared. Cohort A (2009–2010) completed simulation-based training at commencement of internship, receiving individually coached practice on the LP simulator until achieving a predefined mastery performance standard. Cohort B (2010–2012) had the same training plus JIPT sessions immediately before their first clinical LP. Main outcome was LP success, defined as obtaining fluid with first needle insertion and <1000 red blood cells per high-power field. Process measures included use of analgesia, early stylet removal, and overall attempts. RESULTS: A total of 436 first infant LPs were analyzed. The LP success rate in cohort A was 35% (13/37), compared with 38% (152/399) in cohort B (95% confidence interval for difference [CI diff], −15% to +18%). Cohort B exhibited greater analgesia use (68% vs 19%; 95% CI diff, 33% to 59%), early stylet removal (69% vs 54%; 95% CI diff, 0% to 32%), and lower mean number of attempts (1.4 ± 0.6 vs 2.1 ± 1.6, P < .01) compared with cohort A. CONCLUSIONS: Across multiple institutions, intern success rates with infant LP are poor. Despite improving process measures, adding JIPT to training bundles did not improve success rate. More research is needed on optimal instructional design strategies for infant LP.

Molecular actions of drugs that sensitize cardiac myofilaments to Ca2

Ca 2+ -sensitizers are inotropic agents that modify the response of myofilaments to Ca 2+, and are potentially valuable drugs in the treatment of heart failure. These agents have diverse chemical structures, and in some cases also have effects as inhibitors of phosphodiesterase activity. Advantages of their actions include vasodilation combined with inotropic effects. Reduction in the amounts of Ca 2+ required to activate the myofilaments also lowers the oxygen consumption required for Ca 2+ transport, lowers the threat of arrhythmias, and may blunt Ca 2+ -dependent transcriptional and translational mechanisms leading to hypertrophy and failure. Although diastolic abnormalities and impaired relaxation were thought to be potential undesirable effects of Ca 2+ -sensitizers, studies of hearts beating in situ indicate that this may not be a major problem. We focus here on Ca 2+ -sensitizers that act on cardiac troponin C, the Ca 2+ receptor that triggers activation of the actin-myosin interaction. Structural studies have identified a unique mode of Ca 2+ signaling in cardiac troponin C that should aid in targeting drugs to the heart. Moreover, identification of docking sites of Ca 2+ -sensitizers on troponin C suggest new directions for rational drug design.

Altered signaling surrounding the C-lobe of cardiac troponin C in myofilaments containing an α-tropomyosin mutation linked to familial hypertrophic cardiomyopathy

A region of interaction between the near N-terminal of cardiac troponin I (cTnI) and the C-lobe of troponin C (cTnC), where troponin T (cTnT) binds, appears to be critical in regulation of myofilament Ca(2+)-activation. We probed whether functional consequences of modulation of this interface influence the function of tropomyosin (Tm) in thin filament activation. We modified the C-lobe of cTnC directly by addition of the Ca(2+)-sensitizer, EMD 57033, and indirectly by replacing native cTnI with cTnI-containing Glu residues at Ser-43 and Ser-45 (cTnI-S43E/S45E) in myofilaments from hearts of non-transgenic (NTG) and transgenic (TG) mice expressing a point mutation on alpha-Tm (E180G) linked to familial hypertrophic cardiomyopathy. Introduction of cTnI-S43E/S45E induced a significantly greater reduction in tension in TG myofilaments compared to NTG controls. Furthermore, the effect of EMD 57033 to restore Ca(2+)-sensitivity was higher in TG compared to NTG fiber bundles containing cTnI-S43E/S45E and compared to TG or NTG fiber bundles containing native TnI. Our results indicate that alterations in regions of interaction among the N-terminal of cTnI, the C-lobe of cTnC, and the C-terminus of cTnT are important in the regulation of myofilament activity. Although levels of phosphorylation at protein kinase C-dependent sites were the same in TG and NTG myofilaments, our data indicate that the effects of phosphorylation were more depressive in TG hearts.

Modulation of Thin Filament Activity in Long and Short Term Regulation of Cardiac Function

The contraction and relaxation of heart muscle is exquisitely controlled to match the venous return to the cardiac output. If the control mechanisms work, they ensure that output of the right or left ventricle occurs over a broad range with little change in end diastolic volume (EDV). If the control mechanisms fail and increases in cardiac output occur with relatively large changes in EDV, then homeostasis is threatened by edema and by relatively inefficient conversion of wall tension into pressure. Moreover, chronic elevations in EDV and the associated stretch of the myocardium engage pathways for hypertrophic signaling. With hypertrophic adaptation, homeostasis is restored, but this is likely to be temporary; with continued strain on the cells eventually maladapation occurs and a viscous cycle of cardiac cell growth and depressed function leads to the syndrome of heart failure.