Divya Pathak
Postdoctoral Scholar, Biochemistry
https://orcid.org/0000-0001-9944-2308All Publications
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Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.
Proceedings of the National Academy of Sciences of the United States of America
2024; 121 (19): e2318413121
Abstract
Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.
View details for DOI 10.1073/pnas.2318413121
View details for PubMedID 38683993
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Cryo-EM structure of the folded-back state of human β-cardiac myosin.
Nature communications
2023; 14 (1): 3166
Abstract
To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human β-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.6 Å resolution, providing details of all the interfaces stabilizing the 'off' state. The structure shows that these interfaces are hot spots of hypertrophic cardiomyopathy mutations that are thought to cause hypercontractility by destabilizing the 'off' state. Importantly, the cardiac and smooth muscle myosin IHM structures dramatically differ, providing structural evidence for the divergent physiological regulation of these muscle types. The cardiac IHM structure will facilitate development of clinically useful new molecules that modulate IHM stability.
View details for DOI 10.1038/s41467-023-38698-w
View details for PubMedID 37258552
View details for PubMedCentralID 3156359
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Cryo-EM structure of the folded-back state of human β-cardiac myosin.
bioRxiv : the preprint server for biology
2023
Abstract
During normal levels of exertion, many cardiac muscle myosin heads are sequestered in an off-state even during systolic contraction to save energy and for precise regulation. They can be converted to an on-state when exertion is increased. Hypercontractility caused by hypertrophic cardiomyopathy (HCM) myosin mutations is often the result of shifting the equilibrium toward more heads in the on-state. The off-state is equated with a folded-back structure known as the interacting head motif (IHM), which is a regulatory feature of all muscle myosins and class-2 non-muscle myosins. We report here the human β-cardiac myosin IHM structure to 3.6 Å resolution. The structure shows that the interfaces are hot spots of HCM mutations and reveals details of the significant interactions. Importantly, the structures of cardiac and smooth muscle myosin IHMs are dramatically different. This challenges the concept that the IHM structure is conserved in all muscle types and opens new perspectives in the understanding of muscle physiology. The cardiac IHM structure has been the missing puzzle piece to fully understand the development of inherited cardiomyopathies. This work will pave the way for the development of new molecules able to stabilize or destabilize the IHM in a personalized medicine approach. *This manuscript was submitted to Nature Communications in August 2022 and dealt efficiently by the editors. All reviewers received this version of the manuscript before 9 208 August 2022. They also received coordinates and maps of our high resolution structure on the 18 208 August 2022. Due to slowness of at least one reviewer, this contribution was delayed for acceptance by Nature Communications and we are now depositing in bioRxiv the originally submitted version written in July 2022 for everyone to see. Indeed, two bioRxiv contributions at lower resolution but adding similar concepts on thick filament regulation were deposited this week in bioRxiv, one of the contributions having had access to our coordinates. We hope that our data at high resolution will be helpful for all readers that appreciate that high resolution information is required to build accurate atomic models and discuss implications for sarcomere regulation and the effects of cardiomyopathy mutations on heart muscle function.
View details for DOI 10.1101/2023.04.15.536999
View details for PubMedID 37131793
View details for PubMedCentralID PMC10153137
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Cryo-EM structure of the folded-back state of human beta-cardiac myosin
CELL PRESS. 2023: 258A-259A
View details for Web of Science ID 000989629701377
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Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction.
Biophysical journal
2023; 122 (3S1): 147a
View details for DOI 10.1016/j.bpj.2022.11.1011
View details for PubMedID 36782676
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Cryo-EM structure of the folded-back state of human beta-cardiac myosin.
Biophysical journal
2023; 122 (3S1): 258a-259a
View details for DOI 10.1016/j.bpj.2022.11.1489
View details for PubMedID 36783267
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Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction
CELL PRESS. 2023: 147A
View details for Web of Science ID 000989629700719
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Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function.
eLife
2022; 11
Abstract
Mutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of β-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.
View details for DOI 10.7554/eLife.76805
View details for PubMedID 35767336
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Understanding the molecular basis of HCM-causing mutations in cardiac myosin and cardiac myosin binding protein-C
CELL PRESS. 2022: 255A
View details for Web of Science ID 000759523001505
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Patient Perspectives of Inpatient Telemedicine During COVID-19: A Qualitative Assessment.
JMIR formative research
2022
Abstract
Telemedicine has been adopted in the inpatient setting to facilitate clinical interactions between on-site clinicians and isolated hospitalized patients. Such remote interactions have the potential to reduce pathogen exposure and use of personal protective equipment but may also pose new safety concerns given prior evidence that isolated patients can receive suboptimal care. Formal evaluations into the use and practical acceptance of inpatient telemedicine amongst hospitalized patients are lacking.We aimed to evaluate the experience of patients hospitalized for COVID-19 with inpatient telemedicine introduced as an infection control measure during the pandemic.We conducted a qualitative evaluation in a COVID-19 designated non-intensive care hospital unit at a large academic health center (Stanford Health Care) October 2020 through January 2021. Semi-structured qualitative interviews focused on patient experience, impact on quality of care, communication, and mental health. Purposive sampling were used to recruit participants represent-ing diversity across varying demographics until thematic saturation was reached. Interview transcripts were qualitatively analyzed using an inductive-deductive approach.Interviews with 20 hospitalized patients suggested non-emergency clinical care and bridging to in-person care comprised the majority of inpatient telemedicine use. Nurses were reported to enter the room and call on the tablet far more frequently than physicians, who typically entered the room at least daily. Patients reported broad acceptance of the technology, citing improved convenience and reduced anxiety but preferred in-person care where possible. Quality of care was believed to be similar to in-person care with the exception of a few patients who wanted more frequent in-person examinations. Ongoing challenges included low volume, shifting tablet location, and inconsistent verbal introductions from the clinical team.Patient experiences with in-patient telemedicine were largely favorable. Although most patients ex-pressed a preference for in-person care, telemedicine was acceptable given the circumstances asso-ciated with COVID-19. Technical and care team use improvements may enhance acceptability. Fur-ther evaluation is needed to understand the impact of inpatient telemedicine and the optimal balance between in-person and virtual care in the hospital setting.
View details for DOI 10.2196/32933
View details for PubMedID 35147510
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Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.
ACS nano
2021
Abstract
Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.
View details for DOI 10.1021/acsnano.1c02242
View details for PubMedID 34060810
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Lipidomics Suggests a New Role for Ceramide Synthase in Phagocytosis
ACS CHEMICAL BIOLOGY
2018; 13 (8): 2280-2287
Abstract
Phagocytosis is an evolutionarily conserved biological process where pathogens or cellular debris are cleared by engulfing them in a membrane-enclosed cellular compartment called the phagosome. The formation, maturation, and subsequent degradation of a phagosome is an important immune response essential for protection against many pathogens. Yet, the global lipid profile of phagosomes remains unknown, especially as a function of their maturation in immune cells. Here, we show using mass spectrometry based quantitative lipidomics that the ceramide class of lipids, especially very long chain ceramides, are enriched on maturing phagosomes with a concomitant decrease in the biosynthetic precursors of ceramides. We thus posit a new function for the enzyme ceramide synthase during phagocytosis in mammalian macrophages. Biochemical assays, cellular lipid feeding experiments, and pharmacological blockade of ceramide synthase together show that this enzyme indeed controls the flux of ceramides on maturing phagosomes. We also find similar results in the primitive eukaryote Dictyostelium discoideum, suggesting that ceramide enrichment may be evolutionarily conserved and likely an indispensible step in phagosome maturation.
View details for DOI 10.1021/acschembio.8b00438
View details for Web of Science ID 000442452300045
View details for PubMedID 29963848
View details for PubMedCentralID PMC6102644
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Lipid - Motor Interactions: Soap Opera or Symphony?
CURRENT OPINION IN CELL BIOLOGY
2017; 44: 79-85
Abstract
Intracellular transport of organelles can be driven by multiple motor proteins that bind to the lipid membrane of the organelle and work as a team. We review present knowledge on how lipids orchestrate the recruitment of motors to a membrane. Looking beyond recruitment, we also discuss how heterogeneity and local mechanical properties of the membrane may influence function of motor-teams. These issues gain importance because phagocytosed pathogens use lipid-centric strategies to manipulate motors and survive in host cells.
View details for DOI 10.1016/j.ceb.2016.09.005
View details for Web of Science ID 000400022400012
View details for PubMedID 27697416
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Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes
CELL
2016; 164 (4): 722-734
Abstract
Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.
View details for DOI 10.1016/j.cell.2015.12.054
View details for Web of Science ID 000369998300017
View details for PubMedID 26853472
View details for PubMedCentralID PMC4752818