Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, ΔK210. We determined the molecular mechanism of ΔK210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that ΔK210 not only reduces contractility but also causes cellular hypertrophy and impairs cardiomyocytes’ ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results help link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.

27Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a 28 major indicator for heart transplant. The disease is frequently caused by mutations of 29 sarcomeric proteins; however, it is not well understood how these molecular mutations 30 lead to alterations in cellular organization and contractility. To address this critical gap in 31 our knowledge, we studied the molecular and cellular consequences of a DCM mutation 32 in troponin-T, DK210. We determined the molecular mechanism of DK210 and used 33 computational modeling to predict that the mutation should reduce the force per 34 sarcomere. In mutant cardiomyocytes, we found that DK210 not only reduces contractility, 35 but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to 36 changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and 37 disease). These results link the molecular and cellular phenotypes and implicate 38 alterations in mechanosensing as an important factor in the development of DCM. 39Results 100 DK210 decreases calcium sensitivity in an in vitro motility assay 101We set out to decipher the molecular mechanism of the DK210 mutation in vitro. 102The molecular effects of cardiomyopathy mutations depend on the myosin isoform (7-9, 103 35-37) and therefore, we used porcine cardiac ventricular myosin (38). Porcine ventricular 104 cardiac myosin (MYH7) is 97% identical to human, while murine cardiac myosin (MYH6) 105 is only 92% identical. Porcine cardiac myosin has very similar biophysical properties to 106 human cardiac myosin, including the kinetics of the ATPase cycle, step size, and 107 sensitivity to load (38-41), making it an ideal myosin for biophysical studies. 108Given the role of troponin-T in thin filament regulation, we first determined whether 109 the DK210 mutation affects calcium-based regulation of myosin binding to thin filaments 110 using an in vitro motility assay (42). Reconstituted thin filaments, consisting of porcine 111 cardiac actin and recombinantly expressed human troponin and tropomyosin, were added 112 to a flow cell coated with porcine cardiac myosin in the presence of ATP. The speed of 113 filament translocation was measured as a function of added calcium. As has been 114 reported previously, the speed of regulated thin filament translocation increased 115 sigmoidally with increasing Ca 2+ concentration (43), ( Figure 1B). Data were fit with the Hill 116 equation to obtain the pCa50 (i.e., the concentration of calcium necessary for half-117 maximal activation). Consistent with previous studies using mouse cardiac, rabbit cardiac, 118 and rabbit skeletal muscle fibers (31, 33, 44), DK210 shows a right-shifted curve (pCa50 119 = 5.7 ± 0.1) compared to the WT (pCa50 = 6.1 ± 0.1; p < 0.0001), meaning more calcium 120 is needed for the same level of activation. This suggests that the mutant could show 121 impaired force production during a calcium transient. 122 7 123 Molecular mechanism of DK210-induced changes in thin filament regulat...

The Drosophila leg imaginal disc provides a paradigm with which to understand the fundamental developmental mechanisms that generate an intricate appendage structure. Leg formation depends on the subdivision of the leg proximodistal (PD) axis into broad domains by the leg gap genes. The leg gap genes act combinatorially to initiate the expression of the Notch ligands Delta (Dl) and Serrate (Ser) in a segmental pattern. Dl and Ser induce the expression of a set of transcriptional regulators along the segment border, which mediate leg segment growth and joint morphogenesis. Here we show that Lines accumulates in nuclei in the presumptive tarsus and the inter-joints of proximal leg segments and governs the formation of these structures by destabilizing the nuclear protein Bowl. Across the presumptive tarsus, lines modulates the opposing expression landscapes of the leg gap gene dachshund (dac) and the tarsal PD genes, bric-a-brac 2 (bab), apterous (ap) and BarH1 (Bar). In this manner, lines inhibits proximal tarsal fates and promotes medial and distal tarsal fates. Across proximal leg segments, lines antagonizes bowl to promote Dl expression by relief-of-repression. In turn, Dl signals asymmetrically to stabilize Bowl in adjacent distal cells. Bowl, then, acts cell-autonomously, together with one or more redundant factors, to repress Dl expression. Together, lines and bowl act as a binary switch to generate a stable Notch signaling interface between Dl-expressing cells and adjacent distal cell. lines plays analogous roles in developing antennae, which are serially homologous to legs, suggesting evolutionarily conserved roles for lines in ventral appendage formation.

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.

The Drosophila leg imaginal disc consists of a peripheral region that contributes to adult body wall, and a central region that forms the leg proper. While the patterning signals and transcription factors that determine the identity of adult structures have been identified, the mechanisms that determine the shape of these structures remain largely unknown. The family of Rho GTPases, which consists of 7 members in flies, modulates cell adhesion, actomyosin contractility, protrusive membrane activity, and cell-matrix adhesion to generate mechanical forces that shape adult structures. The Rho GTPases are ubiquitously expressed and it remains unclear how they orchestrate morphogenetic events. The Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase activating proteins (RhoGAPs), which respectively activate and deactivate corresponding Rho GTPases, have been proposed to regulate the activity of Rho signaling cascades in specific spatiotemporal patterns to orchestrate morphogenetic events. Here we identify restricted expression of 12 of the 20 RhoGEFs and 10 of the 22 Rho RhoGAPs encoded in Drosophila during metamorphosis. Expression of a subset of each family of RhoGTPase regulators was restricted to motile cell populations including tendon, muscle, trachea, and peripodial stalk cells. A second subset was restricted either to all presumptive joints or only to presumptive tarsal joints. Depletion of individual RhoGEFs and RhoGAPs in the epithelium of the disc proper identified several joint-specific genes, which act downstream of segmental patterning signals to control epithelial morphogenesis. Our studies provide a framework with which to understand how Rho signaling cascades orchestrate complex morphogenetic events in multicellular organisms, and evidence that patterning signals regulate these cascades to control apical constriction and epithelial invagination at presumptive joints.

Central to embryonic development is the generation of molecular asymmetries across fields of undifferentiated cells. The Drosophila wing imaginal disc provides a powerful system with which to understand how such asymmetries are generated and how they contribute to formation of a complex structure. Early in development, the wing primordium is subdivided into a thin layer of peripodial epithelium (PE) and an apposing thickened layer of pseudostratified columnar epithelium (CE), known as the disc proper (DP). The DP gives rise to the wing blade, hinge and dorsal mesothorax, whereas the PE makes only a minor contribution to the ventral hinge and pleura. The mechanisms that generate this major asymmetry and its contribution to wing development are poorly understood. The Lines protein destabilizes the nuclear protein Bowl in ectodermal structures. Here, we show that Bowl accumulates in the PE from early stages of wing development and is absent from the DP. Broad inhibition of Bowl in the PE resulted in the replacement of the PE with a mirror image duplication of the DP. The failure to generate the PE severely compromised wing growth and the formation of the notum. Conversely, the activation of bowl in the DP (by removal or inhibition of lines function) resulted in the transformation of the DP into PE. Thus, we provide evidence that bowl and lines act as a binary switch to subdivide the wing primordium into PE and DP, and assign crucial roles for this asymmetry in wing growth and patterning.

25Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in 26 the sarcomere and generate pulling forces. This process is regulated by calcium, and it 27 can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations 28 (e.g., b-adrenergic stimulation), and pharmacological interventions. Therefore, it is 29 important to have a methodology to robustly determine the mechanism of these 30 perturbations and statistically evaluate their effects. Here, we present an approach to 31 measure the equilibrium constants that govern muscle activation, estimate uncertainty in 32 these parameters, and statistically test the effects of perturbations. We provide a 33 MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials 34 that make this approach accessible. The hypothesis testing and error estimation 35 approaches described here are broadly applicable, and the provided tools work with other 36 types of data, including cellular measurements. To demonstrate the utility of the 37 approach, we apply it to determine the biophysical mechanism of a mutation that causes 38 familial hypertrophic cardiomyopathy. This approach is generally useful for studying the 39 mechanisms of muscle diseases and therapeutic interventions that target muscle 40 contraction. 41 42

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least 6 of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 milliseconds of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin’s binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 mM vs. 0.36 mM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets.