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Circulation May 2024Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants...
BACKGROUND
Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored.
METHODS
We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human allelic pathogenic SNVs.
RESULTS
Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy.
CONCLUSIONS
Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.
PubMed: 38752340
DOI: 10.1161/CIRCULATIONAHA.123.067890 -
Biologics : Targets & Therapy 2024Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived...
Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.
BACKGROUND AND OBJECTIVES
Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively).
METHODS
For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations.
RESULTS
Pan-cardiomyocyte marker, including troponin T2 () and alpha-actinin-2 (), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (), myosin light chain 7 (), and K+ channel Kir3.4 (), and iPS-VM-specific markers, ie, gap junction α-1 (), myosin light chain 2 (), and alpha-1-subunit of a voltage-dependent L-type calcium channel (), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development.
CONCLUSION
IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.
PubMed: 38746773
DOI: 10.2147/BTT.S448054 -
BioRxiv : the Preprint Server For... May 2024During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin...
UNLABELLED
During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure.
SIGNIFICANCE STATEMENT
Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.
PubMed: 38746225
DOI: 10.1101/2024.04.30.591979 -
The Journal of Clinical Investigation May 2024Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using...
Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.
PubMed: 38743494
DOI: 10.1172/JCI174527 -
Scientific Reports May 2024Biomaterial scaffolds play a pivotal role in the advancement of cultured meat technology, facilitating essential processes like cell attachment, growth, specialization,...
Global transcriptome profiles provide insights into muscle cell development and differentiation on microstructured marine biopolymer scaffolds for cultured meat production.
Biomaterial scaffolds play a pivotal role in the advancement of cultured meat technology, facilitating essential processes like cell attachment, growth, specialization, and alignment. Currently, there exists limited knowledge concerning the creation of consumable scaffolds tailored for cultured meat applications. This investigation aimed to produce edible scaffolds featuring both smooth and patterned surfaces, utilizing biomaterials such as salmon gelatin, alginate, agarose and glycerol, pertinent to cultured meat and adhering to food safety protocols. The primary objective of this research was to uncover variations in transcriptomes profiles between flat and microstructured edible scaffolds fabricated from marine-derived biopolymers, leveraging high-throughput sequencing techniques. Expression analysis revealed noteworthy disparities in transcriptome profiles when comparing the flat and microstructured scaffold configurations against a control condition. Employing gene functional enrichment analysis for the microstructured versus flat scaffold conditions yielded substantial enrichment ratios, highlighting pertinent gene modules linked to the development of skeletal muscle. Notable functional aspects included filament sliding, muscle contraction, and the organization of sarcomeres. By shedding light on these intricate processes, this study offers insights into the fundamental mechanisms underpinning the generation of muscle-specific cultured meat.
Topics: Transcriptome; Tissue Scaffolds; Animals; Biopolymers; Cell Differentiation; Meat; Muscle Development; Alginates; Gene Expression Profiling; Sepharose; Biocompatible Materials; Gelatin; Muscle Cells; Salmon; In Vitro Meat
PubMed: 38740842
DOI: 10.1038/s41598-024-61458-9 -
Biophysical Reviews Apr 2024[This corrects the article DOI: 10.1007/s12551-021-00840-7.].
[This corrects the article DOI: 10.1007/s12551-021-00840-7.].
PubMed: 38737206
DOI: 10.1007/s12551-023-01086-1 -
International Journal of Molecular... Apr 2024Mutations in the gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal...
Mutations in the gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal skeletal muscles and has a variable age of onset and disease severity. This variability has been attributed to genetic background differences among individuals; however, such variants have not been well characterized. To identify such variants, we investigated a multigeneration family in which affected individuals are diagnosed with LGMD1B. The primary genetic cause of LGMD1B in this family is a dominant mutation that activates a cryptic splice site, leading to a five-nucleotide deletion in the mature mRNA. This results in a frame shift and a premature stop in translation. Skeletal muscle biopsies from the family members showed dystrophic features of variable severity, with the muscle fibers of some family members possessing cores, regions of sarcomeric disruption, and a paucity of mitochondria, not commonly associated with LGMD1B. Using whole genome sequencing (WGS), we identified 21 DNA sequence variants that segregate with the family members possessing more profound dystrophic features and muscle cores. These include a relatively common variant in (). This variant was given priority because another mutation in causes autosomal dominant centronuclear myopathy-4, which causes cores in addition to centrally positioned nuclei. Therefore, we analyzed muscle biopsies from family members and discovered that those with both the mutation and the variant contain muscle cores that accumulated both CCDC78 and RyR1. Muscle cores containing mislocalized CCDC78 and RyR1 were absent in the less profoundly affected family members possessing only the mutation. Taken together, our findings suggest that a relatively common variant in can impart profound muscle pathology in combination with a mutation and accounts for variability in skeletal muscle disease phenotypes.
Topics: Adult; Female; Humans; Male; Middle Aged; Lamin Type A; Muscle Proteins; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Mutation; Pedigree; Microtubule-Associated Proteins
PubMed: 38732148
DOI: 10.3390/ijms25094930 -
Journal of Medicinal Chemistry May 2024Cardiac myosin activation has been shown to be a viable approach for the treatment of heart failure with reduced ejection fraction. Here, we report the discovery of...
Cardiac myosin activation has been shown to be a viable approach for the treatment of heart failure with reduced ejection fraction. Here, we report the discovery of nelutroctiv (), a selective cardiac troponin activator intended for patients with cardiovascular conditions where cardiac contractility is reduced. Discovery of nelutroctiv began with a high-throughput screen that identified compound , a muscle selective cardiac sarcomere activator devoid of phosphodiesterase-3 activity. Optimization of druglike properties for led to the replacement of the sulfonamide and aniline substituents which resulted in improved pharmacokinetic (PK) profiles and a reduced potential for human drug-drug interactions. echocardiography assessment of the optimized leads showed concentration dependent increases in fractional shortening and an improved pharmacodynamic window compared to myosin activator . Overall, nelutroctiv was found to possess the desired selectivity, a favorable pharmacodynamic window relative to myosin activators, and a preclinical PK profile to support clinical development.
Topics: Humans; Animals; Myocardial Contraction; Cardiovascular Diseases; Rats; Structure-Activity Relationship; Male; Drug Discovery; Troponin; Mice; Rats, Sprague-Dawley; Sulfonamides
PubMed: 38729623
DOI: 10.1021/acs.jmedchem.3c02413 -
Journal of Biomechanics May 2024Connective tissues can be recognized as an important structural support element in muscles. Recent studies have also highlighted its importance in active force...
Connective tissues can be recognized as an important structural support element in muscles. Recent studies have also highlighted its importance in active force generation and transmission between muscles, particularly through the epimysium. In the present study, we aimed to investigate the impact of the endomysium, the connective tissue surrounding muscle fibers, on both passive and active force production. Pairs of skeletal muscle fibers were extracted from the extensor digitorum longus muscles of rats and, after chemical skinning, their passive and active force-length relationships were measured under two conditions: (i) with the endomysium between muscle fibers intact, and (ii) after its dissection. We found that the dissection of the endomysium caused force to significantly decrease in both active (by 22.2 % when normalized to the maximum isometric force; p < 0.001) and passive conditions (by 25.9 % when normalized to the maximum isometric force; p = 0.034). These findings indicate that the absence of endomysium compromises muscle fiber's not only passive but also active force production. This effect may be attributed to increased heterogeneity in sarcomere lengths, enhanced lattice spacing between myofilaments, or a diminished role of trans-sarcolemmal proteins due to dissecting the endomysium. Future investigations into the underlying mechanisms and their implications for various extracellular matrix-related diseases are warranted.
Topics: Animals; Rats; Muscle Fibers, Skeletal; Rats, Wistar; Connective Tissue; Sarcomeres; Male; Muscle, Skeletal; Biomechanical Phenomena; Isometric Contraction; Muscle Contraction
PubMed: 38723428
DOI: 10.1016/j.jbiomech.2024.112134 -
Circulation Jun 2024The "2024 AHA/ACC/AMSSM/HRS/PACES/SCMR Guideline for the Management of Hypertrophic Cardiomyopathy" provides recommendations to guide clinicians in the management of...
2024 AHA/ACC/AMSSM/HRS/PACES/SCMR Guideline for the Management of Hypertrophic Cardiomyopathy: A Report of the American Heart Association/American College of Cardiology Joint Committee on Clinical Practice Guidelines.
AIM
The "2024 AHA/ACC/AMSSM/HRS/PACES/SCMR Guideline for the Management of Hypertrophic Cardiomyopathy" provides recommendations to guide clinicians in the management of patients with hypertrophic cardiomyopathy.
METHODS
A comprehensive literature search was conducted from September 14, 2022, to November 22, 2022, encompassing studies, reviews, and other evidence on human subjects that were published in English from PubMed, EMBASE, the Cochrane Library, the Agency for Healthcare Research and Quality, and other selected databases relevant to this guideline. Additional relevant studies, published through May 23, 2023, during the guideline writing process, were also considered by the writing committee and added to the evidence tables, where appropriate.
STRUCTURE
Hypertrophic cardiomyopathy remains a common genetic heart disease reported in populations globally. Recommendations from the "2020 AHA/ACC Guideline for the Diagnosis and Treatment of Patients With Hypertrophic Cardiomyopathy" have been updated with new evidence to guide clinicians.
Topics: Humans; American Heart Association; Cardiology; Cardiomyopathy, Hypertrophic; Disease Management; United States
PubMed: 38718139
DOI: 10.1161/CIR.0000000000001250