Julius Bogomolovas

Filamin C (FLNC) is an essential cytoskeletal protein required for cardiomyocyte structural integrity and mechanotransduction. Truncating FLNC variants cause haploinsufficiency associated with dilated and arrhythmogenic cardiomyopathies, yet no therapies currently address the underlying defect of FLNC deficiency. Gene replacement is challenging because the approximately 8.2-kb FLNC coding sequence exceeds the packaging capacity of conventional adeno-associated virus (AAV) vectors. Here, we engineered a triple-AAV strategy optimized for full-length FLNC reconstitution in the heart. Screening in HEK293 cells identified GP41-1 and IMPDH as a compatible orthogonal pair for efficient full-length FLNC reconstitution. In tamoxifen-inducible, cardiomyocyte-specific Flnc knockout mice, systemic delivery of optimized MyoAAV2A vectors restored full-length FLNC expression and re-established sarcomeric localization following cardiomyocyte FLNC depletion. A total vector dose of 1 × 10¹³ genome copies/kg restored FLNC to approximately endogenous abundance, preserved systolic function and ventricular dimensions, limited cardiac enlargement, attenuated fibrotic remodeling and prevented premature death. FLNC replacement also attenuated pathological transcriptional remodeling and compensatory changes in cytoskeletal and junction-associated proteins. These findings demonstrate that systemic split-intein-mediated reconstitution of full-length FLNC can preserve cardiac structure and function in vivo, providing preclinical support for a mutation-independent therapeutic strategy for FLNC-associated cardiomyopathy.

Protein kinase N2 (PKN2) is essential for embryonic heart development, but the precise timing and cellular requirement in cardiomyocytes are not well understood. Here we show that, in a constitutive cardiomyocyte-specific knockout (cKO) of PKN2, we observe partially penetrant early postnatal lethality, non-progressive systolic dysfunction in survivors, and a distinctive “coin-pouch” ventricular geometry resembling the embryonic shape, linking the phenotype to a developmental origin rather than ongoing pathology. By tracking heart morphogenesis, we find that mutant and control hearts are still grossly indistinguishable at E10.5, defining a stage prior to overt morphological divergence. Integrative RNA-seq, proteomics, and phosphoproteomics at E10.5 reveal transcriptional and proteomic signatures consistent with induction of actin cytoskeleton and motility-associated programs alongside repression of chromosome-condensation and mitotic modules in PKN2 cKO hearts. These molecular changes coincide with reduced cardiomyocyte proliferation at E10.5–E11.5 and early divergence in ventricular-shape trajectories. Quantitative light-sheet microscopy–based morphometrics of inducible cardiomyocyte-specific PKN2 knockout hearts pinpoints the critical dependence window: tamoxifen administration at E7.5 reproduces the ventricular geometry characteristic of constitutive PKN2 cKO hearts, whereas administration at E10.5 spares gross morphology. Together, these data show that cardiomyocytes depend on PKN2 during an early proliferative phase, characterized by cytoskeletal remodeling and motility-associated programs, that contributes to ventricular wall formation, after which PKN2 becomes dispensable for gross ventricular chamber morphogenesis. This temporally restricted requirement reconciles previous observations of PKN2’s essential role in mesodermal and epithelial morphogenesis, suggesting a shared function in coordinating cytoskeletal remodeling with tissue expansion during organogenesis.

Pathogenic variants in ALPK3 (α-protein kinase 3), an atypical α‑kinase acting as a sarcomeric M-band scaffold, cause cardiomyopathy with severity linked to zygosity. We present a comprehensive review with systematic curation of peer-reviewed clinical and experimental reports through June 9, 2025, encompassing 156 patient-level variants and all published preclinical models. Biallelic loss-of-function variants lead to severe, often lethal cardiomyopathy with prenatal or early onset presentation and extracardiac involvement. Heterozygous protein-truncating variants, defined as nonsense or frameshift (resulting from insertion/deletion events or splicing mutations), explain ≈1% to 4% of adult hypertrophic cardiomyopathy, often with apical/septal hypertrophy, right ventricular involvement, fibrosis, and risk of progression. ALPK3 lacks catalytic activity and maintains sarcomeric proteostasis by scaffolding MYOMs (myomesins), MuRF (muscle ring-finger protein) E3 ligases, and SQSTM1 (sequestosome-1)/p62. Loss of this scaffolding function displaces MYOMs, drives thick‑filament protein aggregation, and precipitates severe contractile dysfunction in human induced pluripotent stem cell–derived cardiomyocytes and multiple mouse models. Therapeutic proof‑of‑concept has now been achieved on 2 fronts: (1) pharmacological correction of sarcomeric hypercontractility with the myosin inhibitor mavacamten and (2) durable phenotypic rescue in global knockout mice using an adeno-associated virus–delivered miniALPK3 gene‑replacement construct. Together, these data position ALPK3 cardiomyopathy as a compelling target for precision medicine. Early genetic diagnosis, genotype-tailored surveillance, and focused development of gene-replacement or editing strategies, potentially combined with modulators of the ALPK3-MuRF proteostatic axis, offer a realistic path to disease-modifying therapy for this once enigmatic condition.

Background: ALPK3 (α protein kinase 3) is an atypical kinase highly expressed in human and murine hearts. Biallelic loss‐of‐function mutations in ALPK3 lead to pediatric cardiomyopathy. The specific stages at which ALPK3 is essential for cardiac function and the mechanisms by which it regulates cardiac function require further exploration. Methods and Results: We generated ALPK3 global knockout and inducible cardiac‐specific knockout mice. We performed time‐course physiological and morphological assessments to determine ALPK3's role in neonatal and adult hearts. We also generated an Alpk3 ‐3xFLAG‐HA knock‐in mouse model to determine endogenous ALPK3 localization. To investigate mechanisms of ALPK3 regulation, we performed biochemical assays and RNA sequencing experiments in global knockout mice. ALPK3 is critical for both neonatal and adult cardiac function. Loss of ALPK3 at germline and adult stages leads to dilated cardiomyopathy. Approximately 75% of germline ALPK3 mice die within 1 month, while surviving mutant mice develop dilated cardiomyopathy that transitions to left ventricular hypertrophy, mirroring clinical manifestations in human patients with biallelic ALPK3 mutations. We found that ALPK3 localizes to the M‐band in both neonatal and adult cardiomyocytes and interacts with muscle RING‐finger proteins, which may regulate thick filament protein turnover. Conclusions: Our study highlights the necessity of ALPK3 in neonatal and adult cardiac function. Our data support a model in which ALPK3 serves as a scaffold protein to recruit machineries essential for regulating thick filament protein turnover.

Protein kinases play crucial roles in regulating cellular processes, making real-time visualization of their activity essential for understanding signaling dynamics. While genetically encoded fluorescent biosensors have emerged as powerful tools for studying kinase activity, their development for many kinases remains challenging due to the lack of suitable substrate peptides. Here, we present a novel approach for identifying peptide substrates and demonstrate its effectiveness by developing a biosensor for Protein Kinase N (PKN) activity. Our method identified a new PKN substrate peptide that we optimized for use in a fluorescent biosensor design. The resulting biosensor shows specificity for PKN family kinases and can detect both overexpressed and endogenous PKN activity in live cells. Importantly, our biosensor revealed sustained basal PKN2 activity at the plasma membrane, identifying it as a PKN2 activity hotspot. This work not only provides a valuable tool for studying PKN signaling but also demonstrates a promising strategy for developing biosensors for other understudied kinases, potentially expanding our ability to monitor kinase activity across the human kinome.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI , Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of β myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity’s role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

The use of digital image analysis and count regression models contributes to the reproducibility and rigor of histological studies in cardiovascular research. The use of formalized computer-based quantification strategies of histological images essentially removes potential researcher bias, allows for higher analysis throughput, and enables easy sharing of formalized quantification tools, contributing to research transparency, and data transferability. Moreover, the use of count regression models rather than ratios in statistical analysis of cell population data incorporates the extent of sampling into analysis and acknowledges the non-Gaussian nature of count distributions. Using quantification of proliferating cardiomyocytes in embryonic murine hearts as an example, we describe how these improvements can be implemented using open-source artificial intelligence-based image analysis tools and novel count regression models to efficiently analyze real-life data.

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction (NJ) of skeletal muscles. The major MG autoantigen is nicotinic acetylcholine receptor. Other autoantigens at the NJ include MuSK, LRP4 and agrin. Autoantibodies to the intra-sarcomeric striated muscle-specific gigantic protein titin, although not directed to the NJ, are invaluable biomarkers for thymoma and MG disease severity. Thymus and thymoma are critical in MG mechanisms and management. Titin autoantibodies bind to a 30 KDa titin segment, the main immunogenic region (MIR), consisting of an Ig-FnIII-FnIII 3-domain tandem, termed I109–I111. In this work, we further resolved the localization of titin epitope(s) to facilitate the development of more specific anti-titin diagnostics. For this, we expressed protein samples corresponding to 8 MIR and non-MIR titin fragments and tested 77 anti-titin sera for antibody binding using ELISA, competition experiments and Western blots. All anti-MIR antibodies were bound exclusively to the central MIR domain, I110, and to its containing titin segments. Most antibodies were bound also to SDS-denatured I110 on Western blots, suggesting that their epitope(s) are non-conformational. No significant difference was observed between thymoma and non-thymoma patients or between early- and late-onset MG. In addition, atomic 3D-structures of the MIR and its subcomponents were elucidated using X-ray crystallography. These immunological and structural data will allow further studies into the atomic determinants underlying titin-based autoimmunity, improved diagnostics and how to eventually treat titin autoimmunity associated co-morbidities.