Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions

Background: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, TTN functions and therapeutic targets. Methods: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Finally, we engineered a full-length TTN protein reporter assay and utilized next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. Results: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR-ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome-editing using SpCas9 and a TTNtv-specific guide RNA restored TTN protein reading frame, which increased full length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. Conclusions: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. While both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a "one-and-done" genome editing strategy to target ∼30% of DCM-associated TTNtvs.

In vivo potentiation of post-tetanic twitch across age and sex

Twitch force potentiation of fast-twitch skeletal muscle is produced by repetitive stimulation that can be achieved from either (1) the staircase effect (continual low frequency stimulation) or (2) post-tetanic potentiation (a 1–2 s high-frequency tetanic stimulation). Previous studies examining twitch force potentiation have been conducted in vitro and shown that it is related to phosphorylation of myosin regulatory light chain (pRLC). We previously found, in vitro, reduced potentiation of twitch force and decreased pRLC in ovariectomized (Ovx, estrogen-deficient) compared with sham-operated (estrogen-replete) mice. Thus, we questioned whether this phenomenon occurred in vivo and whether age and sex would affect the potentiation of twitch force. Using an in vivo post-tetanic potentiation method (one twitch contraction followed by a tetanic contraction—100 Hz for 1,000 ms with 0.01 ms pulses, and two post-tetanic twitch contractions), we investigated twitch torque potentiation in C57BL/6 young and old, male and female mice. There were significant main effects of sex (P < 0.001) and age (P < 0.001) on body mass and significant main effects of sex (P < 0.001) on tibialis anterior and extensor digitorum longus muscle masses, with males and aged being relatively greater. Analysis of twitch torque using a three-way ANOVA across time, age, and sex showed a significant main effect of time (pre < post; P < 0.001), time × age (P = 0.038), and time × sex (P = 0.028), indicating potentiation occurred in young and old, males and females. Analysis of twitch torque potentiation (percent increase) using a two-way ANOVA revealed a significant main effect of age (young = 45.16 ± 2.04 versus old = 27.88 ± 9.96; P < 0.001) with no effect of sex (P = 0.215). In summary, enhanced generation of twitch force of skeletal muscle using a post-tetanic potentiation method does occur in vivo and is affected by age but not sex, as there is greater twitch torque potentiation in young than old mice.

Sarcolemmal Excitability, M-Wave Changes, and Conduction Velocity During a Sustained Low-Force Contraction

This study was undertaken to investigate whether sarcolemmal excitability is impaired during a sustained low-force contraction [10% maximal voluntary contraction (MVC)] by assessing muscle conduction velocity and also by analyzing separately the first and second phases of the muscle compound action potential (M wave). Twenty-one participants sustained an isometric knee extension of 10% MVC for 3min. M waves were evoked by supramaximal single shocks to the femoral nerve given at 10-s intervals. The amplitude, duration, and area of the first and second M-wave phases were computed. Muscle fiber conduction velocity, voluntary surface electromyographic (EMG), perceived effort, MVC force, peak twitch force, and temperature were also recorded. The main findings were: (1) During the sustained contraction, conduction velocity remained unchanged. (2) The amplitude of the M-wave first phase decreased for the first ~30s (−7%, p<0.05) and stabilized thereafter, whereas the second phase amplitude increased for the initial ~30s (+7%, p<0.05), before stabilizing. (3) Both duration and area decreased steeply during the first ~30s, and then more gradually for the rest of the contraction. (4) During the sustained contraction, perceived effort increased fivefold, whereas knee extension EMG increased by ~10%. (5) Maximal voluntary force and peak twitch force decreased (respectively, −9% and −10%, p<0.05) after the low-force contraction. Collectively, the present results indicate that sarcolemmal excitability is well preserved during a sustained 10% MVC task. A depression of the M-wave first phase during a low-force contraction can occur even in the absence of changes in membrane excitability. The development of fatigue during a low-force contraction can occur without alteration of membrane excitability.

Recovery From Eccentric Squat Exercise in Resistance-Trained Young and Master Athletes With Similar Maximum Strength: Combining Cold Water Immersion and Compression

The aim of this study was to investigate whether recovery from eccentric squat exercise varies depending on age and to assess whether the use of a mixed-method recovery (MMR) consisting of cold water immersion and compression tights benefits recovery. Sixteen healthy and resistance-trained young (age, 22.1±2.1years; N=8) and master male athletes (age, 52.4±3.5years; N=8), who had a similar half squat 1-repetition maximum relative to body weight, completed two identical squat exercise training sessions, separated by a 2-week washout period. Training sessions were followed by either MMR or passive recovery (PR). Internal training loads [heart rate and blood lactate concentration (BLa)] were recorded during and after squat sessions. Furthermore, maximal voluntary isometric contraction (MVIC) force, countermovement jump (CMJ) height, resting twitch force of the knee extensors, serum concentration of creatine kinase (CK), muscle soreness (MS), and perceived physical performance capability (PPC) were determined before and after training as well as after 24, 48, and 72h of recovery. A three-way mixed ANOVA revealed a significant time effect of the squat protocol on markers of fatigue and recovery (p<0.05; decreased MVIC, CMJ, twitch force, and PPC; increased CK and MS). Age-related differences were found for BLa, MS, and PPC (higher post-exercise fatigue in younger athletes). A significant two-way interaction between recovery strategy and time of measurement was found for MS and PPC (p<0.05; faster recovery after MMR). In three participants (two young and one master athlete), the individual results revealed a consistently positive response to MMR. In conclusion, master athletes neither reach higher fatigue levels nor recover more slowly than the younger athletes. Furthermore, the results indicate that MMR after resistance exercise does not contribute to a faster recovery of physical performance, neuromuscular function, or muscle damage, but promotes recovery of perceptual measures regardless of age.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

Abstract P491: Myosin-7 E848G Mutation Induces P53-associated Cell Death That Leads To Impaired Tissue Contractility In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy

Introduction: Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and occasional impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood. Hypothesis: We previously identified a novel MYH7 E848G mutation associated with HCM. We hypothesize E848G induces cell death that results in impaired tissue contractility in a dose-dependent manner. Methods: We created MYH7 expressing CMs with WT/WT, E848G/WT, or E848G/E848G alleles by CRISPR/Cas9 gene-editing patient-specific induced pluripotent stem cells (hiPSCs). hiPSC-derived cardiomyocytes were metabolically purified and cocultured with stromal cells on PDMS posts to create 3D engineered heart tissues (EHTs) or cultured as a monolayer. Results: Day 65 monolayer E848G/E848G CMs had 48.5% effective cell number relative to WT/WT. p53 (2.80 ± .11-fold), p21 (7.24 ± .18-fold), and BAX (1.64 ± 0.14-fold) mRNA transcripts were upregulated in day 60 monolayer E848G/E848G relative to WT/WT. E848G/E848G EHTs (n = 12) exhibited lower maximum active twitch force (104.6 ± 18.2 μN) and smaller 2D projected area (5.31 ± 0.22 mm 2 ) at day 14 relative to WT/WT (n = 15; 238.0 ± 20.4 μN; 6.87 ± 0.26 mm 2 ). E848G/WT EHTs (n = 7) had intermediate twitch force (168.7 ± 12.2 μN) and 2D area (6.18 ± 0.36 mm 2 ). Conclusion: These results suggest the MYH7 E848G mutation induces p53-associated cell death that leads to reduced tissue contractility. Ongoing studies will elucidate the molecular mechanism through which E848G activates cell death pathways. Figure: Representative EHTs. L-R: WT/WT, E848G/WT, E848G/E848G.

Perimenopausal women show modulation of excitatory and inhibitory neuromuscular mechanisms

Background Menopausal transition exposes women to an early decline in muscle force and motor function. Changes in muscle quality and function, especially in lower limbs, are crucial, as they expose individuals to increased risk of falls. To elucidate some of the related neuromuscular mechanisms, we investigated cortical inhibition and peripheral muscle twitch force potentiation in women during the early and late stages of perimenopause. Methods Participants were 63 women aged 48–55 years categorized as early (EP, n = 25) or late (LP, n = 38) perimenopausal according to serum follicle-stimulating hormone (FSH) levels and menstrual diaries. EP women had an irregular menstrual cycle and FSH < 25 IU/L, while LP women had an irregular cycle and > 25 IU/L. We examined motor evoked potential (MEP) and silent period (SP) elicited by transcranial magnetic stimulation (TMS), in the tibialis anterior muscle at 20%, 40%, and 60% of maximal voluntary contraction (MVC) levels, and twitch force potentiation in plantar flexors. Results EP group showed a longer SP duration in 40% MVC condition and larger motor evoked potential amplitude in 20% MVC condition compared to the LP group. No group difference was detected in twitch force potentiation; however, it correlated negatively with FSH levels. Other factors, such as age, height, body mass index, or physical activity did not explain group differences. Conclusions Our preliminary results indicate subtle modulation in both TMS-induced inhibitory and excitatory mechanisms and twitch force potentiation in women already in the late perimenopausal stage. This suggests that the reduction of estrogens may have an accelerating role in the aging process of neuromuscular control.

Paxilline Prevents the Onset of Myotonic Stiffness in Pharmacologically Induced Myotonia: A Preclinical Investigation

Reduced Cl− conductance causes inhibited muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. This represents the pathomechanism of myotonia congenita. Due to the prevailing data suggesting that an increased potassium level is a main contributor, we studied the effect of a modulator of a big conductance Ca2+- and voltage-activated K+ channels (BK) modulator on contraction and relaxation of slow- and high-twitch muscle specimen before and after the pharmacological induction of myotonia. Human and murine muscle specimens (wild-type and BK−/−) were exposed to anthracene-9-carboxylic acid (9-AC) to inhibit CLC-1 chloride channels and to induce myotonia in-vitro. Functional effects of BK-channel activation and blockade were investigated by exposing slow-twitch (soleus) and fast-twitch (extensor digitorum longus) murine muscle specimens or human musculus vastus lateralis to an activator (NS1608) and a blocker (Paxilline), respectively. Muscle-twitch force and relaxation times (T90/10) were monitored. Compared to wild type, fast-twitch muscle specimen of BK−/− mice resulted in a significantly decreased T90/10 in presence of 9-AC. Paxilline significantly shortened T90/10 of murine slow- and fast-twitch muscles as well as human vastus lateralis muscle. Moreover, twitch force was significantly reduced after application of Paxilline in myotonic muscle. NS1608 had opposite effects to Paxilline and aggravated the onset of myotonic activity by prolongation of T90/10. The currently used standard therapy for myotonia is, in some individuals, not very effective. This in vitro study demonstrated that a BK channel blocker lowers myotonic stiffness and thus highlights its potential therapeutic option in myotonia congenital (MC).

Effect of New Zealand Blackcurrant Extract on Isometric Contraction-Induced Fatigue and Recovery: Potential Muscle-Fiber Specific Effects

New Zealand blackcurrant (NZBC) extract has shown performance-enhancing effects during cycling, running and sport climbing. We examined effects of NZBC extract on (1) voluntary and twitch force of the quadriceps femoris muscles during repeated isometric contraction-induced fatigue, (2) twitch force during recovery and (3) muscle fiber-specific effects. Familiarized recreationally active males (n = 12, age: 24 ± 5 yrs; height: 180 ± 5 cm; body mass: 89 ± 11 kg) performed sixteen, 5-s voluntary maximal isometric contractions (iMVC) separated by 3-s rest. Twitch force was recorded before, during the 3-s rests and 5-min recovery. Supplementation consisted of 7-days intake of NZBC extract (600 mg∙day−1 containing 210 mg anthocyanin) in a double-blind, randomized, placebo-controlled crossover design with a 14-days washout. NZBC extract allowed for greater force in the first quartile of the iMVCs. Twitch force at baseline was 12% higher with NZBC extract (p = 0.05). However, there was no effect of NZBC for twitch force during the 16-iMVCs and recovery. Based on the maximum post-activation potentiation during the placebo 16-iMVCs, four subjects were classified of having a predominant type I or II muscle fiber typology. In type II, NZBC extract provided a trend for increased MVC force (~14%) in the first quartile and for type I in the fourth quartile (~10%). In type I, NZBC extract seemed to have higher twitch forces during the fatiguing exercise protocol and recovery, indicating increased fatigue resistance. New Zealand blackcurrant extract affects force during repeated maximal isometric contractions. Future work on mechanisms by NZBC extract for muscle fiber-specific fatigue-induced force responses is warranted.