Biology subjects

McNamara, J.

Publications and source records attributed to McNamara, J..

6 recordsLinked to original sources

bioRxiv2025

Alpha Protein Kinase 3 Gene Therapy Restores Heart Function in Mouse and Human Models of Cardiomyopathy

Truncating variants in the Alpha Kinase 3 (ALPK3) gene have recently emerged as an important cause of genetic cardiomyopathy globally and here we demonstrate the efficacy and safety of a viral-based gene replacement therapy for ALPK3. Given the loss-of-function nature of these variants, we reasoned that a gene replacement approach would improve heart function in this patient population. We demonstrate that the delivery of full length ALPK3 via adeno-associated virus could restore contractile function in human cardiac organoids and in vivo mouse models carrying clinically relevant mutations in ALPK3. The role of disrupted proteostasis networks in multiple forms of genetic cardiomyopathy suggest that delivery this novel AAV-ALPK3 may provide functional benefit outside of cardiomyopathy induced by ALPK3. Titin truncating variants (TTNtv) are the most common cause of dilated cardiomyopathy, and interestingly also contributes to the M-Band protein quality control network coordinated by ALPK3. Notably, in human cardiac organoids carrying a TTNtv we observed that the ALPK3 gene therapy could completely restore contractile deficits. This opens the exciting prospect for indication expansion of AAV-ALPK3 into other forms of cardiomyopathy that currently have no therapeutic options.

physiology↗

bioRxiv2024

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids

BACKGROUNDMYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. C_LIO_LIThe MYBPC3D389V-mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. C_LIO_LIOur data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure

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 StatementRecently, 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.

physiology↗

bioRxiv2023

Affinity purification-mass spectrometry and single fiber physiology/proteomics reveals mechanistic insights of C18ORF25

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in skeletal muscle following acute exercise in humans. Phosphorylation was shown to improve ex vivo skeletal muscle contractile function in mice but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein:protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, and revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localised to the nucleus and the contractile apparatus in skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled detailed assessment of various steps in the excitation-contraction (EC) pathway including SR calcium handling and force generation followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein:phenotype associations and 36 fiber-type specific differences following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

physiology↗

bioRxiv2023

Unlocking the Role of sMyBP-C: A Key Player in Skeletal Muscle Development and Growth

Skeletal muscle is the largest organ in the body, responsible for gross movement and metabolic regulation. Recently, variants in the MYBPC1 gene have been implicated in a variety of developmental muscle diseases, such as distal arthrogryposis. How MYBPC1 variants cause disease is not well understood. Here, through a collection of novel gene-edited mouse models, we define a critical role for slow myosin binding protein-C (sMyBP-C), encoded by MYBPC1, across muscle development, growth, and maintenance during prenatal, perinatal, postnatal and adult stages. Specifically, Mybpc1 knockout mice exhibited early postnatal lethality and impaired skeletal muscle formation and structure, skeletal deformity, and respiratory failure. Moreover, a conditional knockout of Mybpc1 in perinatal, postnatal and adult stages demonstrates impaired postnatal muscle growth and function secondary to disrupted actomyosin interaction and sarcomere structural integrity. These findings confirm the essential role of sMyBP-C in skeletal muscle and reveal specific functions in both prenatal embryonic musculoskeletal development and postnatal muscle growth and function.

physiology↗

bioRxiv2018

Clinical implications of identifying pathogenic variants in aortic dissection patients with whole exome sequencing

BackgroundThoracic aortic dissection is an emergent life-threatening condition. Routine screening for genetic variants causing thoracic aortic dissection is not currently performed for patients or their family members.\n\nMethodsWe performed whole exome sequencing of 240 patients with thoracic aortic dissection (n=235) or rupture (n=5) and 258 controls matched for age, sex, and ancestry. Blinded to case-control status, we annotated variants in 11 genes for pathogenicity.\n\nResultsTwenty-four pathogenic variants in 6 genes (COL3A1, FBN1, LOX, PRKG1, SMAD3, TGFBR2) were identified in 26 individuals, representing 10.8% of aortic cases and 0% of controls. Among dissection cases, we compared those with pathogenic variants to those without and found that pathogenic variant carriers had significantly earlier onset of dissection (41 vs. 57 years), higher rates of root aneurysm (54% vs. 30%), less hypertension (15% vs. 57%), lower rates of smoking (19% vs. 45%), and greater incidence of aortic disease in family members. Multivariable logistic regression showed significant risk factors associated with pathogenic variants are age <50 [odds ratio (OR) = 5.5; 95% CI: 1.6-19.7], no history of hypertension (OR=5.6; 95% CI: 1.4-22.3) and family history of aortic disease (mother: OR=5.7; 95% CI: 1.4-22.3, siblings: OR=5.1; 95% CI 1.1-23.9, children: OR=6.0; 95% CI: 1.4-26.7).\n\nConclusionsClinical genetic testing of known hereditary thoracic aortic dissection genes should be considered in patients with aortic dissection, followed by cascade screening of family members, especially in patients with age-of-onset of aortic dissection <50 years old, family history of aortic disease, and no history of hypertension.

genetics↗