Biology subjects

Toepfer, C. N.

Publications and source records attributed to Toepfer, C. N..

5 recordsLinked to original sources

bioRxiv2026

Unmasking Supervillin: SVIL haploinsufficiency causes hypertrophic cardiomyopathy by impairing mechanotransduction and cellular energetics

BackgroundRare heterozygous loss-of-function (LoF) variants in SVIL, encoding the Z-disk and costameric protein supervillin, have recently been identified as a cause of hypertrophic cardiomyopathy (HCM). Although supervillin is implicated in actin-dependent mechanotransduction, the mechanisms linking SVIL deficiency to cardiomyopathy remain poorly understood. Homozygous LoF cause a novel skeletal Myofibrillar Myopathy-10 (MFM-10) while heterozygous LoF cause HCM without skeletal myopathy. In this study we use a human model system to disentangle the LoF pathomechanism of the scaffolding protein supervillin in cardiomyocytes and its clinical implications. MethodsUsing CRISPR/Cas-9 we engineered a representative pathogenic LoF variant Q255X into an isogenic induced pluripotent stem cell (iPSC) line creating the heterozygous SVILQ255X/+ and homozygous SVILQ255X/Q255X cell lines. These lines were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) and cellular phenotypes were assessed using bulk RNA-sequencing, LC-MS proteomics, electrophysiological and calcium handling analyses, contractility measurements, sarcomere organization analysis, Seahorse metabolic flux assay, and pharmacological intervention with mavacamten. ResultsThe Q255X variant resulted in SVIL haploinsufficiency at both RNA and protein levels with no evidence of a truncated protein. Compared with isogenic controls, SVILQ255X/+ iPSC-CMs demonstrated action potential shortening, calcium transient elongation, sarcomeric disorganization and hypertrophy, and impaired mitochondrial respiration. Multi-omic analyses of SVILQ255X/+ iPSC-CMs showed a profile of cellular stress and inflammation, hypertrophic and pro-fibrotic signalling, and a pseudohypoxic state driven by decreased respiration and a HIF-induced glycolytic shift. These abnormalities were not present in SVILQ255X/Q255X cardiomyocytes, consistent with a relatively limited cardiac phenotype reported in homozygous variant carriers. Mavacamten improved sarcomeric disorganization and hypertrophy in SVILQ255X/+ cells but did not rescue energetic compromise. ConclusionsPathogenic heterozygous SVIL LoF produces a distinct cellular phenotype characterized by impaired mechanotransduction, mitochondrial dysfunction, and maladaptive metabolic remodelling that promotes hypertrophic and pro-fibrotic signalling. These findings define a mechanistic basis for SVIL-associated cardiomyopathy and identify metabolic dysfunction as a potential therapeutic target beyond sarcomere-directed therapy. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISVIL haploinsufficiency causes HCM through a mechanism distinct from canonical sarcomeric disease, characterized by impaired mechanotransduction, mitochondrial dysfunction, and pseudohypoxia-driven metabolic remodeling. C_LIO_LIHeterozygous SVIL loss of function produces a substantially more severe cardiomyocyte phenotype than homozygous loss of function, providing a mechanistic explanation for the predominance of cardiac disease in heterozygous variant carriers. C_LIO_LIMavacamten improves sarcomeric organization but does not restore impaired mitochondrial respiration, demonstrating that energetic dysfunction persists despite sarcomere-directed therapy. C_LI What Are the Clinical Implications?O_LIOur findings give functional evidence to support SVIL as a clinically relevant HCM disease gene and its inclusion in clinical genetic testing panels. C_LIO_LIThese findings establish SVIL-associated cardiomyopathy as a mechanistically distinct form of HCM and offer insight into the pathomechanism of Z-disk and costameric HCM C_LIO_LIThe persistence of mitochondrial dysfunction despite myosin inhibition suggests that drugs targeting mitochondrial bioenergetics may be a therapeutic strategy in patients with SVIL-associated cardiomyopathy. C_LI

cell biology↗

bioRxiv2025

Targeting Troponin I Phosphorylation for Precision Treatment of Diastolic Dysfunction

BackgroundDefective cardiac relaxation (diastolic dysfunction) is prevalent in heart failure, particularly in the context of diabetes, obesity, hypertension, and ageing, and is associated with increased mortality. Yet there is no available treatment that directly targets this abnormal relaxation. Phosphorylation of troponin I by cAMP-dependent protein kinase A (PKA) promotes relaxation, but global PKA activation has widespread, undesirable effects. Here we investigated whether selectively enhancing troponin I phosphorylation could improve relaxation without engaging the broader PKA signalling network. MethodsTo resolve cAMP signalling at subcellular scales, we combined real-time cAMP measurements with FRET-based genetically encoded reporters targeted to defined nanodomains, alongside biochemical and genetic approaches. Protein-protein interaction surfaces were mapped using peptide-array technology, which guided the design of a disruptor peptide to displace specific interactions. The peptides effects on cardiomyocyte function were assessed in vitro using rodent and human cardiomyocytes with biochemical assays, real-time imaging, and work-loop analysis. In vivo effects were evaluated through echocardiography and haemodynamic measurements. ResultsWe identified a previously unrecognized regulatory mechanism in which the cAMP-hydrolyzing enzyme PDE4D9 interacts with troponin I and restricts local cAMP levels within a nanometre-scale domain, thereby selectively controlling troponin I phosphorylation. We found that PDE4D9-troponin I association is markedly increased in cardiac disease in both rodents and humans. A peptide that displaces PDE4D9 from troponin I in a mouse model of heart failure selectively enhances troponin I phosphorylation, accelerates cardiomyocyte relaxation, and prevents diastolic dysfunction, without compromising systolic contractility. ConclusionsThese findings reveal a highly specific regulatory nanodomain governing troponin I phosphorylation and points to a first-in-class, mechanism-based therapeutic strategy with the potential to address a major unmet contributor to heart failure. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe identify a previously unrecognized, nanometre-scale regulatory mechanism in which PDE4D9 binds troponin I to locally suppress cAMP, limit troponin I phosphorylation and attenuate relaxation. C_LIO_LIWe demonstrate that this mechanism becomes maladaptive in disease: PDE4D9-troponin I interaction is markedly upregulated in failing hearts in both rodents and humans, which can mechanistically explain the impaired cardiac relaxation. C_LIO_LIWe show that this interaction is druggable: a rationally designed peptide can selectively displace PDE4D9, restore localized cAMP-PKA signalling, and normalize myocardial relaxation without global cAMP perturbation. C_LIO_LIDisplacement of PDE4D9 from TPNI improves relaxation with no impact on the calcium transient amplitude and without adversely affecting systolic output and cardiac reserve. C_LI What Are the Clinical Implications?O_LIDiastolic dysfunction, a major cause of heart failure with no targeted treatments, may be amenable to therapies that modulate cAMP signalling with subcellular precision rather than global pathway modulation. C_LIO_LISelective disruption of PDE4D9-troponin I binding offers a mechanism-based approach with potential to restore diastolic function while minimizing off-target effects typical of GPCR-directed therapies. C_LIO_LIBy enhancing relaxation while preserving systolic function and cardiac reserve, this approach may overcome limitations of myofilament-targeted therapies currently under evaluation for diastolic dysfunction that exhibit negative inotropic effects. C_LIO_LIMore broadly, this work establishes a platform for targeting localized signalling domains, a concept that may extend beyond cardiovascular disease to other pathological conditions in which dysregulated cAMP contributes to dysfunction. C_LI

cell biology↗

bioRxiv2024

An ALPK3 truncation variant causing autosomal dominant hypertrophic cardiomyopathy is partially rescued by mavacamten

The ALPK3 gene encodes alpha-protein kinase 3, a cardiac pseudo-kinase of unknown function. Heterozygous truncating variants (ALPK3tv) can cause dominant adult-onset Hypertrophic Cardiomyopathy (HCM). Here we confirm an excess of ALPK3tv in sarcomere-gene negative HCM patients. Moreover, we generated a novel knock-in mouse model carrying an ALPK3tv (K201X). Homozygous animals displayed hypertrophy and systolic dysfunction. Heterozygous animals demonstrated no obvious baseline; however, they had an aggravated hypertrophic response upon chronic adrenergic challenge. Isolated cardiomyocytes from heterozygous and homozygous mice showed reduced basal sarcomere length with prolonged relaxation, whilst calcium transients showed increased diastolic calcium levels and prolonged re-uptake. We also observed reduced fractional shortening. Protein kinase A-mediated phosphorylation, including that of cardiac troponin I, was significantly decreased. In agreement with the cellular HCM phenotype, reduced ratios of myosin heads in the super-relaxed state were measured. Contractile and calcium handling defects were partly corrected by treatment with mavacamten, a novel myosin inhibitor. For the first time with a non-sarcomere HCM variant, we have demonstrated hallmark changes in cardiac contractility and calcium handling, some resembling sarcomere-positive HCM, while others are distinct and DCM-like. Mavacamten is able to partially rescue the cellular phenotype, hence could be beneficial to HCM patients with ALPK3tv.

genetics↗

bioRxiv2022

Human iPSC-CMs and in-silico technologies define mechanisms and accelerate targeted pharmacogenetics in hypertrophic cardiomyopathy

Cardiomyopathies have unresolved genotype-phenotype relationships and lack disease-specific treatments. Here we identify genotype-specific pathomechanisms and therapeutic targets combining experimental hiPSC-CM modelling and human-based cardiac electromechanical in-silico modelling and simulation bridging from specific mutations to clinical biomarkers. We select hypertrophic cardiomyopathy as a challenge for this approach and study genetic variations that mutate proteins of the thick (MYH7R403Q/+) and thin filaments (TNNT2R92Q/+, TNNI3R21C/+) of the cardiac sarcomere. We show that destabilisation of myosin super relaxation drives disease in MYH7R403Q/+ with secondary effects on thin filament activation, which are corrected by Mavacamten. Thin filament variants TNNT2R92Q/+ and TNNI3R21C/+ share calcium regulation-related pathomechanisms, for which Mavacamten provides incomplete salvage. We define the ideal characteristics of a novel thin filament-targeting compound and show its efficacy in-silico. We demonstrate that hybrid human-based hiPSC-CM and in-silico studies accelerate pathomechanism discovery and classification testing, improving clinical interpretation of genetic variants, and directing rational therapeutic targeting and design.

bioengineering↗

bioRxiv2018

MYBPC3 Mutations cause Hypertrophic Cardiomyopathy by Dysregulating Myosin: Implications for Therapy

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin binding protein-C; cMyBPC) or myosin missense mutations cause hyper-contractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches we explored how depletion of cMyBPC altered sarcomere function. We demonstrate that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM) normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation - enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across all phases of the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in DCM patients, while inhibiting myosin by MYK-461 should benefit the substantial proportion of HCM patients with MYBPC3 mutations.\n\nOne Sentence SummaryAnalyses of cardiomyocytes with hypertrophic cardiomyopathy mutations in MYBPC3 reveal that these directly activate myosin contraction by disrupting myosin states of relaxation, and that genetic or pharmacological manipulation of myosin therapeutically abates the effects of MYBPC3 mutations.

genetics↗