Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the Ca V 1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying Ca V 1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tonecued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.utism and autism spectrum disorder (ASD) are characterized by the concomitant occurrence of impaired social interaction; restricted, perseverative, and stereotypical behavior; and abnormal communication skills (1). However, the etiology remains largely unknown, in large part because most cases of ASD arise from a mixture of multiple environmental and multiple genetic influences (2), making it difficult to forge causal links to behavior. In the face of such complexity, insights might be gleaned from simple forms of ASD, generated by single, highly penetrant mutations. Timothy syndrome (TS), is a rare disorder strongly associated with autism or ASD (penetrance w75%; P = 1.2 × 10 −8 ). Other symptoms of TS include long QT syndrome, webbed fingers and toes, dysmorphic facial features, and immunodeficiency (3). Importantly, Splawski et al. (3) traced the disease to a single nucleotide mutation in the gene encoding the pore-forming subunit of an L-type calcium channel (Ca V 1.2). This sporadic glycine-to-arginine mutation is located at position 406 in exon 8A [Splawski's terminology (3), VNDAV-coding exon, low (w20%) expression in brain and heart]. If a Gly-toArg mutation occurs in the more highly (w80%) expressed (4) alternative exon 8 (MQDAM-coding exon, 59 of exon 8A) it causes a more severe variant of TS (TS2). In heterologous expression ...

Key points Skeletal muscle fatigue limits performance in various physical activities, with exercise intolerance being a key symptom in a broad spectrum of diseases.We investigated whether a small molecule fast skeletal troponin activator (FSTA), CK‐2066260, can mitigate muscle fatigue by reducing the cytosolic free [Ca2+] required to produce a given submaximal force and hence decreasing the energy requirement.Isolated intact single mouse muscle fibres and rat muscles in‐situ treated with CK‐2066260 showed improved muscle endurance., which was accompanied by decreased ATP demand and reduced glycogen usage.CK‐2066260 treatment improved in‐vivo exercise capacity in healthy rats and in a rat model of peripheral artery insufficiency.In conclusion, we show that the FSTA CK‐2066260 effectively counteracts muscle fatigue in rodent skeletal muscle in vitro, in situ, and in vivo. This may translate to humans and provide a promising pharmacological treatment to patients suffering from severe muscle weakness and exercise intolerance. AbstractSkeletal muscle fatigue limits performance during physical exercise and exacerbated muscle fatigue is a prominent symptom among a broad spectrum of diseases. The present study investigated whether skeletal muscle fatigue is affected by the fast skeletal muscle troponin activator (FSTA) CK‐2066260, which increases myofibrillar Ca2+ sensitivity and amplifies the submaximal force response. Because more force is produced for a given Ca2+, we hypothesized that CK‐2066260 could mitigate muscle fatigue by reducing the energetic cost of muscle activation. Isolated single mouse muscle fibres were fatigued by 100 repeated 350 ms contractions while measuring force and the cytosolic free [Ca2+] or [Mg2+] ([Mg2+]i). When starting fatiguing stimulation at matching forces (i.e. lower stimulation frequency with CK‐2066260): force was decreased by ∼50% with and by ∼75% without CK‐2066260; [Mg2+]i was increased by ∼10% with and ∼32% without CK‐2066260, reflecting a larger decrease in [ATP] in the latter. The glycogen content in in situ stimulated rat muscles fatigued by repeated contractions at matching forces was about two times higher with than without CK‐2066260. Voluntary exercise capacity, assessed by rats performing rotarod exercise and treadmill running, was improved in the presence of CK‐2066260. CK‐2066260 treatment also increased skeletal muscle fatigue resistance and exercise performance in a rat model of peripheral artery insufficiency. In conclusion, we demonstrate that the FSTA CK‐2066260 mitigates skeletal muscle fatigue by reducing the metabolic cost of force generation.