Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

Rationale: Myofilament length dependent activation (LDA) is the key underlying mechanism of cardiac heterometric autoregulation, commonly referred as the Frank-Starling law of the heart. Although alterations in LDA are common in cardiomyopathic states, the precise structural and biochemical mechanisms underlying LDA remain unknown. Objective: Here, we examine the role of structural changes in the thick filament during diastole, in particular changes in the availability of myosin heads, in determining both calcium sensitivity and maximum contractile force during systole in permeabilized porcine cardiac fibers. Methods and Results: Permeabilized porcine fibers from ventricular myocardium were studied under relaxing conditions at short and long sarcomere length (SL) using muscle mechanics, biochemical measurements, and X-ray diffraction. Upon stretch, porcine myocardium showed the increased calcium sensitivity and maximum calcium activated force characteristic of LDA. Stretch increased diastolic ATP turnover, recruiting reserve myosin heads from the super-relaxed state (SRX) at longer SL. Structurally, X-ray diffraction studies in the relaxed-muscle confirmed a departure from the helical ordering of the thick-filament upon stretch which occurred concomitantly with a displacement of myosin heads towards actin, facilitating cross-bridge formation upon systolic activation. Mavacamten, a selective myosin-motor inhibitor known to weaken the transition to actin-bound power-generating states and to enrich the ordered SRX myosin population, reversed the structural effects of stretch on the thick-filament, blunting the mechanical consequences of stretch; mavacamten did not, however, prevent other structural changes associated with LDA in the sarcomere, such as decreased lattice spacing or troponin-displacement. Conclusions: Our findings strongly indicate that in ventricular muscle, LDA and its systolic consequences are dependent on the population of myosin heads competent to form cross-bridges and involves the recruitment of myosin heads from the reserve SRX pool during diastole.

Background The compromised gut microbiome that results from C-section birth has been hypothesized as a risk factor for the development of non-communicable diseases (NCD). In a double-blind randomized controlled study, 153 infants born by elective C-section received an infant formula supplemented with either synbiotic, prebiotics, or unsupplemented from birth until 4 months old. Vaginally born infants were included as a reference group. Stool samples were collected from day 3 till week 22. Multi-omics were deployed to investigate the impact of mode of delivery and nutrition on the development of the infant gut microbiome, and uncover putative biological mechanisms underlying the role of a compromised microbiome as a risk factor for NCD. Results As early as day 3, infants born vaginally presented a hypoxic and acidic gut environment characterized by an enrichment of strict anaerobes (Bifidobacteriaceae). Infants born by C-section presented the hallmark of a compromised microbiome driven by an enrichment of Enterobacteriaceae. This was associated with meta-omics signatures characteristic of a microbiome adapted to a more oxygen-rich gut environment, enriched with genes associated with reactive oxygen species metabolism and lipopolysaccharide biosynthesis, and depleted in genes involved in the metabolism of milk carbohydrates. The synbiotic formula modulated expression of microbial genes involved in (oligo)saccharide metabolism, which emulates the eco-physiological gut environment observed in vaginally born infants. The resulting hypoxic and acidic milieu prevented the establishment of a compromised microbiome. Conclusions This study deciphers the putative functional hallmarks of a compromised microbiome acquired during C-section birth, and the impact of nutrition that may counteract disturbed microbiome development. Trial registration The study was registered in the Dutch Trial Register (Number: 2838) on 4th April 2011.

Abstract:Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. Previous studies suggest that myosin function can be regulated by entering a super-relaxed state (SRX) with very slow ATP hydrolysis, but the structural determinants of this state are uncertain. Using a combination of biochemical approaches with electron microscopy and X-ray fiber diffraction, we show that the SRX corresponds to a folded-back state of myosin with increased ordering of heads around the thick filament backbone. The small molecule mavacamten induces this conformation, while mutations causing HCM destabilize it. These findings provide a structural basis for an important mode of regulation of cardiac myosin with implications for the pathogenesis of cardiomyopathy and potential therapeutic development.Introduction:

Length dependent activation (LDA) is a property of muscle where increased sarcomere length (SL) leads to increased force of contraction. Despite its key role in both normal and pathological states, the molecular mechanisms underlying LDA are not understood. Previous studies suggest that increased titin-based passive tension at longer SL triggers structural changes in the troponin complex leading to increased Ca 2+ sensitivity. Stretch also appears to release myosin heads from the OFF or super-relaxed state (SRX) increasing the number of available heads for cross-bridge formation, and, hence, maximum force. The small molecule inhibitor of myosin, mavacamten (mava) has been shown to stabilize the SRX state. Here, we used both stretch and mava on permeabilized porcine myocardium for force/pCa measurements coupled with x-ray diffraction to interrogate the mechanisms underlying LDA. Increasing SL from 2.0 to 2.3 um increases Ca 2+ sensitivity and elevates both diastolic and maximal tension (i.e. LDA). Mava abolished LDA while blunting the passive stiffness/tension relationship, suggesting increased compliance. The equatorial x-ray reflection intensity ratio, I 1,1 /I 1,0 , increases with stretch, indicating increased myosin head displacement towards actin. Stretch also decreases the intensities of both the m3 meridional and the MyBPC reflections, suggesting stretch-induced disordering of the thick-filament. Surprisingly, these findings in pig skinned muscle are opposite to those previously reported in intact rat muscle despite robust LDA in both preparations. Consistent with previous studies, stretch increases thick filament strain (indicated by changes in the m6 meridional spacing) and changes in the structure of the troponin complex (indicated by increased intensity of the third order troponin reflection), suggesting that the mechanism for increased Ca 2+ sensitivity are similar in both muscle systems. Interestingly, all diffraction pattern changes that occurred with stretch moved in the opposite direction in the presence of mava. Together, these data indicate that mava is not only capable of stabilizing the SRX state of myosin, but can modulate inter-filament interactions responsible for increasing Ca 2+ sensitivity and force.