Macroautophagy/autophagy is markedly inhibited in the hearts of elderly obese patients with heart failure and preserved ejection fraction (HFpEF). However, the therapeutic relevance and underlying signaling mechanisms of the decline of autophagy in HFpEF remain unclear. We observed that therapeutic nicotinamide adenine dinucleotide (NAD+) repletion via nicotinamide supplementation restores cardioprotective autophagy and mitophagy in preclinical models of obesity-related HFpEF. Targeted and untargeted cardiac acetylome profiling revealed no significant deacetylation of essential autophagy-related proteins, including ATG5, ATG7 and mammalian Atg8-family members (ATG8s), suggesting a SIRT (sirtuin)-independent mechanism of autophagy induction by nicotinamide. Instead, cardiac transcriptomic analysis revealed major shifts in insulin-IGF1 (insulin-like growth factor 1) signaling, a known autophagy inhibitory pathway. Nicotinamide supplementation reverses the HFpEF-associated increase in insulin-IGF1 signaling, whereas exogenous IGF1 counteracts nicotinamide-induced autophagy. Importantly, nicotinamide fails to exert cardioprotective effects in mice lacking the autophagy-related protein ATG5 in cardiomyocytes, implicating autophagy as essential for the therapeutic response. In patients with HFpEF, a metabolic shift diverting nicotinamide away from NAD+ biosynthesis toward catabolism strongly correlates with worsening heart failure and increased cardiovascular mortality, even after adjusting for traditional risk factors. In sum, we demonstrate that NAD+ replenishment improves cardiometabolic HFpEF by restoring cardiac autophagy through suppression of excessive IGF1 signaling.

Cardiomyocyte hypocontractility underlies inherited dilated cardiomyopathy (DCM). Yet, whether fibroblasts modify DCM phenotypes remains unclear despite their regulation of fibrosis, which strongly predicts disease severity. Expression of a hypocontractility-linked sarcomeric variant in mice triggered cardiac fibroblast expansion from the de novo formation of hyperproliferative mechanosensitized fibroblast states, which occurred prior to eccentric myocyte remodeling. Initially, this fibroblast response reorganized fibrillar collagen and stiffened the myocardium, albeit without depositing fibrotic tissue. These adaptations coincided with heightened matrix-integrin receptor interactions and diastolic tension sensation at focal adhesions within fibroblasts. Targeted p38 deletion arrested these cardiac fibroblast responses in DCM mice, which prevented cardiomyocyte remodeling and improved contractility. p38-mediated fibroblast responses were essential regulators of DCM severity, marking a potential cellular target for therapeutic intervention.

Journal Article The ESC Working Group on Myocardial Function Get access Mahmoud Abdellatif, Mahmoud Abdellatif Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, 15 rue de l'Ecole de Médecine, Paris 75006, FranceMetabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, FranceDepartment of Cardiology, Medical University of Graz, Graz, Austria Corresponding authors. Email: [email protected]; Email: [email protected] https://orcid.org/0000-0002-5042-9054 Search for other works by this author on: Oxford Academic PubMed Google Scholar Wolfgang A Linke, Wolfgang A Linke Institute of Physiology II, University of Muenster, Muenster, GermanyClinic for Cardiology and Pneumology, University Medical Center Goettingen, GermanyGerman Centre for Cardiovascular Research (DZHK), Partner site Goettingen, Berlin, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Dana K Dawson, on behalf of Dana K Dawson Aberdeen Cardiovascular and Diabetes Centre, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK Corresponding authors. Email: [email protected]; Email: [email protected] https://orcid.org/0000-0003-2815-4469 Search for other works by this author on: Oxford Academic PubMed Google Scholar The ESC Working Group on Myocardial Function The ESC Working Group on Myocardial Function Search for other works by this author on: Oxford Academic PubMed Google Scholar European Heart Journal, ehae016, https://doi.org/10.1093/eurheartj/ehae016 Published: 18 February 2025

Titin, the largest human protein, serves as the elastic backbone of sarcomeres in muscle cells and imparts passive stiffness to cardiomyocytes. While proteolytic cleavage of elastic titin has been linked to cardiovascular disease, the isolated effects of titin stiffness loss in the living heart remain poorly understood. Here, we developed a knock-in mouse model allowing for the selective cleavage of cardiac titin springs in vivo. Using a time-resolved approach combining MRI, echocardiography, immunofluorescence, and molecular profiling, we demonstrate that titin cleavage does not dilate the heart but induces concentric remodeling, impaired diastolic function, and low-output heart failure. Mechanistically, compromised titin-based restoring forces trigger internal mechanical imbalance and dysconnectivity, which activate fibroblasts and promote extracellular matrix remodeling, revealing titin’s essential role in maintaining cardiac mechanical homeostasis. ### Competing Interest Statement JAK and WAL consult and provide contract research services for multiple biotech companies, but none of these are directly related to the work performed in this study. All other authors declare no competing interests. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, LI 690/14-3 (WAL), LO2951/2-1 (CML) National Institutes of Health, https://ror.org/01cwqze88, HL136737 (JAK), HL172492 (JAK), HL175964 (JAK) Interdisziplinäres Zentrum für Klinische Forschung Münster, , Li1/012/24 (WAL), core unit PIX (CF) German Centre for Cardiovascular Research, https://ror.org/031t5w623, 81X2300190 (WAL, OJM) Medizinerkolleg - Dean of the Medical Faculty Münster, , Stipend 2024-25 (PH)

Progressive myocardial dysfunction in patients with heart failure often involves alterations in myocardial passive stiffness, yet the underlying mechanisms remain incompletely understood. While passive stiffness in the longitudinal direction has been extensively characterized via uniaxial tensile stretching of cardiac specimens, transverse stiffness has received far less attention despite its equal mechanical importance. In this study, we combined atomic force microscopy nanoindentation with stretching assays on myocardial preparations to quantify the relative contributions of the three myofilament networks – actin, myosin, and titin – to passive stiffness in both transverse and longitudinal orientations. We employed a transgenic mouse model in which titin’s elastic springs contain a tobacco etch virus protease (TEVp) recognition site, enabling selective and acute titin cleavage upon TEVp treatment. Actin filaments were severed using a calcium-independent gelsolin fragment, and myosin filaments were dissociated by high-salt extraction. Along the longitudinal axis, titin accounted for over 50

BACKGROUND:Alterations in the buffering of intracellular Ca2+, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca2+ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca2+ and its potential arrhythmogenic role in persAF.METHODS:Transmembrane Ca2+ fluxes (patch-clamp) and intracellular Ca2+ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology were measured on a Langendorff system.RESULTS:Cytosolic Ca2+ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca2+ buffers. In agreement, protein levels of selected Ca2+ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca2+ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca2+ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca2+ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca2+ buffering and atrial arrhythmogenesis.CONCLUSIONS:Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca2+ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca2+ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.

Background How the sarcomeric complex is continuously turned-over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. Methods We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. Results We disprove the prevailing ‘protein pool’ model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age, and that proteolytic extraction is a rate limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. Conclusions Our findings establish a ‘unidirectional replacement’ model for cardiac sarcomeres subunit replacement and identify their turnover principles.

Background: The global rise of obesity and its association with cardiovascular risk factors (CVRF) have highlighted its connection to chronic heart failure (CHF). Paradoxically, obese CHF patients often experience better outcomes, a phenomenon known as the ‘obesity paradox’. This study evaluated the ‘obesity paradox’ within a large cohort in Germany and explored how varying degrees of obesity affect HF outcome. Methods: Anonymized health claims data from the largest German insurer (AOK) for the years 2014–2015 were utilized to analyze 88,247 patients hospitalized for myocardial infarction. This analysis encompassed baseline characteristics, comorbidities, interventions, complications, and long-term outcomes, including overall survival, freedom from CHF, and CHF-related rehospitalization. Patients were categorized based on body mass index. Results: Obese patients encompassed 21.3% of our cohort (median age 68.69 years); they exhibited a higher prevalence of CVRF (p < 0.001) and comorbidities than non-obese patients (median age 70.69 years). Short-term outcomes revealed lower complication rates and mortality (p < 0.001) in obese compared to non-obese patients. Kaplan–Meier estimations for long-term analysis illustrated increased incidences of CHF and rehospitalization rates among the obese, yet with lower overall mortality. Multivariable Cox regression analysis indicated that obese individuals faced a higher risk of developing CHF and being rehospitalized due to CHF but demonstrated better overall survival for those classified as having low-level obesity (p < 0.001). Conclusions: This study underscores favorable short-term outcomes among obese individuals. The ‘obesity paradox’ was confirmed, with more frequent CHF cases and rehospitalizations in the long term, alongside better overall survival for certain degrees of obesity.

To detect the effect of titin stiffness loss on living heart function, we used a mouse model that allows specific, graded cleavage of elastic titin. In this titin-cleavage (TC) mouse, a tobacco etch virus protease (TEVp) recognition site was cloned into elastic titin. Cardiac-specific titin cleavage was achieved by systemically injecting AAV9-TEVp plasmid under a cTnT-promoter; AAV9-eGFP plasmid was injected as a control. Six days post-injection, 22.2 ± 2.9% (N = 3) of cardiac titin were cleaved in heterozygous and 39.8 ± 1.6% (N = 4) in homozygous TC mice. Two weeks post-injection, 30.9 ± 6.1% (N = 3) of titin were cleaved in heterozygous and 57.4 ± 2.1% (N = 5) in homozygous mice. Microscopic analyses of >50% titin-cleaved heart tissue revealed sarcomeres with disrupted or missing I-bands, thinned diameters, and wavy Z-discs; A-bands were preserved longer. Degenerated areas and aggregates were present in cardiomyocytes; fibrosis was abundant. Titin cleavage by 20-30% resulted in Z-disk waviness but little other damage. Western blotting demonstrated increased ubiquitination of cleaved cardiac titin, upregulation of E3 ligases, and autophagy activation, most of which became significant only above ∼50% titin cleavage. Passive force and Ca2+-dependent active force of permeabilized cardiac fiber bundles isolated from >50%-cleaved AAV9-TEVp hearts were significantly reduced compared to control samples. Echocardiography of homozygous, but not heterozygous, TC-Halo mice revealed worsening systolic and diastolic heart function (cardiac output; stroke volume; systolic and diastolic volumes; LV internal diameters; E/E') compared to AAV9-eGFP injected mice, which began to manifest 6 days post-injection and persisted until 14 days post-injection. Mouse voluntary running distance also dropped significantly during this period. Collectively, echocardiographic parameters remained nearly unaltered until ∼55% of titin became cleaved. These findings reveal the important role of titin in cardiac contractility but also a high compensatory potential of the titin-cleaved heart.

Damage of the endothelial glycocalyx (eGC) plays a central role in the development of vascular hyperpermeability and organ damage during systemic inflammation. However, the specific signalling pathways for eGC damage remain poorly defined. Aim of this study was to combine sublingual video-microscopy, plasma proteomics and live cell imaging to uncover further pathways of eGC damage in patients with coronavirus disease 2019 (COVID-19) or bacterial sepsis. This secondary analysis of the prospective multicenter MICROCODE study included 22 patients with COVID-19 and 43 patients with bacterial sepsis admitted to intermediate or intensive care units and 10 healthy controls. Interleukin-6 (IL-6) was strongly associated with damaged eGC and correlated both with eGC dimensions (rs=0.36, p = 0.0015) and circulating eGC biomarkers. In vitro, IL-6 reduced eGC height and coverage, which was inhibited by blocking IL-6 signalling with the anti-IL-6 receptor antibody tocilizumab or the Janus kinase inhibitor tofacitinib. Exposure of endothelial cells to 5

Titin, a spring-like molecule connecting Z-disk and M-band in the half-sarcomere, is thought to contribute to passive force and sarcomeric integrity. Recently, the titin-cleavage (TC) mouse model has been established, where a tobacco etch virus protease (TEVp) cleavage site is cloned into I-band titin, and incubating muscle of TC mice with TEVp allows for I-band titin cleavage. Using this model, we reported that complete titin cleavage reduced passive force in muscle fibre bundles by ∼55%. The remaining force was considered to be related to contribution of other passive elements in muscle fibre.

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when steady-state force is elevated after an active stretch. We investigated titin’s function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-cross-bridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.

The first-in-its-class cardiac drug mavacamten reduces the proportion of so-called ON-state myosin heads in relaxed sarcomeres, altering contraction performance. However, mavacamten is not completely specific to cardiac myosin and can also affect skeletal muscle myosin, an important consideration since mavacamten is administered orally and so will also be present in skeletal tissue. Here, we studied the effect of mavacamten on skeletal muscle structure using small-angle X-ray diffraction. Mavacamten treatment reduced the proportion of ON myosin heads but did not eliminate the molecular underpinnings of length-dependent activation, demonstrating similar effects to those observed in cardiac muscle. These findings provide valuable insights for the potential use of mavacamten as a tool to study muscle contraction across striated muscle.

AbstractIn striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2−/− fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by ~24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

Myocardial passive stiffness is crucial for the heart’s pump function and is determined by mechanical elements, including the extracellular matrix and cytoskeletal filaments; however, their individual contributions are controversially discussed and difficult to quantify. In this study, we targeted the cytoskeletal filaments in a mouse model, which enables the specific, acute and complete cleavage of the sarcomeric titin springs. We show in vitro that each cytoskeletal filament’s stiffness contribution varies depending on whether the elastic or the viscous forces are considered and on strain level. Titin governs myocardial elastic forces, with the largest contribution provided at both low and high strain. Viscous force contributions are more uniformly distributed among the microtubules, titin and actin. The extracellular matrix contributes at high strain. The remaining forces after total target element disruption are likely derived from desmin filaments. Our findings answer longstanding questions about cardiac mechanical architecture and allow better targeting of passive myocardial stiffness in heart failure.