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Coronary heart disease is the leading cause of mortality globally. However, clinical translation of cardioprotective strategies against ischaemic reperfusion injury (IRI) has been disappointing. At The Hatter Cardiovascular Institute, the concept of the Reperfusion Injury Salvage Kinase (RISK) pathway was established, which includes PI3K activation of Akt.1 Our subsequent studies narrowed the target to the alpha isoform of PI3K, PI3Kα, which was shown to be both necessary and sufficient for cardioprotection by ischaemic preconditioning (IPC).2 A molecule that can bypass cell membrane to directly activate PI3Kα would be a promising novel therapy for IRI. As part of a larger collaboration, we recently developed the first allosteric activator of PI3Kα, UCL-TRO-1938 (or ‘1938’), and showed that it significantly reduced infarct size in an in-vivo IRI mouse model.3 We are now investigating the mechanisms of how PI3Kα activation exerts this protection. We hypothesized that 1938 activates PI3Kα signalling in cardiomyocytes, protecting them from injury.In a dose-response experiment in an isolated rat heart IRI model, 10μM 1938 was as protective as IPC, reducing the infarct size from 60 ± 4% to 41 ± 2% (N=8, P =0.01). In a dose-response experiment, in which H9C2 myoblast cells were treated with 500 μM H2O2 for 24 h, 1μM 1938 significantly reduced cell death from 30 ± 2% to 17 ± 1% (n=7, P=0.01). Western blot analysis confirmed that 1938 activates the PI3Kα/Akt pathway in both H9C2 and rat primary cardiomyocytes. After 15 min of treatment 1 μM 1938 significantly increased Akt phosphorylation by 2.0-fold vs. vehicle control (n=4, P=0.003).Future studies include investigating effect of 1938 in cardiomyocyte specific PI3Kα knockout mice to validate our hypothesis, and evaluate 1938’s effectiveness in models with co-morbidities. With comprehensive evaluation of this novel pharmacological approach, we hope to facilitate its successful clinical translation.References Yellon DM, Beikoghli Kalkhoran S, Davidson SM. The RISK pathway leading to mitochondria and cardioprotection: how everything started. Basic Res Cardiol. 2023 May 26;118(1):22. doi: 10.1007/s00395-023-00992-5. PMID: 37233787; PMCID: PMC10220132. Rossello X, Riquelme JA, He Z, Taferner S, Vanhaesebroeck B, Davidson SM, Yellon DM. The role of PI3Kα isoform in cardioprotection. Basic Res Cardiol. 2017 Oct 17;112(6):66. doi: 10.1007/s00395-017-0657-7. PMID: 29043508; PMCID: PMC5645445. Gong GQ, Bilanges B, Allsop B, et al. A small-molecule PI3Kα activator for cardioprotection and neuroregeneration. Nature. 2023 Jun;618(7963):159–168. doi: 10.1038/s41586-023-05972-2. Epub 2023 May 24. PMID: 37225977; PMCID: PMC7614683.

IntroductionAtrial fibrillation (AF) is the most common cause of cardiac arrhythmia globally, highlighting the need for an effective in vitro model to study cardiac physiology and contractility. Investigating human atrial cardiomyocytes in three-dimensional structures will enhance our understanding of their role in atrial physiology and open avenues to explore cell-cell interactions compared to two-dimensional cultures. In this study, we have engineered three-dimensional myocardial microtissues which could offer significant advantages in modelling atrial physiology in vitro and investigating cardiac contractile function and electrophysiology.MethodsHuman induced pluripotent stem cells (iPSCs) were differentiated into ventricular and atrial cardiomyocytes, which were then seeded into non-adherent microwells to form spheroids and into 48 well plates to form monolayers. Spontaneous contractility recordings were performed daily from the onset of cardiomyocyte beating, assessing contraction amplitude, time to peak contraction, and relaxation time. Additionally, calcium transients were recorded to evaluate calcium handling dynamics.ResultsContractility analysis shows that atrial spheroids have significantly shorter contraction duration in comparison to atrial monolayers (259 ± 27 vs. 474 ± 31 ms; p=0.0012, n=4/8), and higher contraction amplitude (444 ± 25 vs. 153 ± 17 a.u.; p<0.0001, n=4/8). Atrial spheroids display a higher spontaneous beating frequency compared to ventricular spheroids (2 ± 0.2497 vs. 0.4 ± 0.0367 Hz; p=0.0037, n=7) and shorter contraction duration after correction for inter-beat interval (289 ± 9 vs. 365 ± 7 ms; p=0.0015, n=4/3). Calcium imaging at 1.5 Hz stimulation reveals chamber-specific differences, with atrial spheroids showing significantly shorter time to calcium peak (67 ± 5 vs. 143 ± 6 ms; p<0.0001, n=5) and shorter calcium transient duration (289 ± 21 vs. 396 ± 11 ms; p=0.0018, n=5).ConclusionOur results reveal distinct electrophysiological and contractile differences between atrial cardiomyocytes cultured in 3D spheroids and 2D monolayers, highlighting the influence of in vitro culture conditions on cellular behaviour. Atrial spheroids provide a more physiologically relevant platform, with an atrial-specific phenotype, offering promising applications for studying cell-cell interactions and their role in AF pathogenesis.

Corrigendum: Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart Wataru Kimura, Feng Xiao, Diana C. Canseco, Shalini Muralidhar, SuWannee Thet, Helen M. Zhang, Yezan Abderrahman, Rui Chen, Joseph A. Garcia, John M. Shelton, James A. Richardson, Abdelrahman M. Ashour, Aroumougame Asaithamby, Hanquan Liang, Chao Xing, Zhigang Lu, Cheng Cheng Zhang & Hesham A. Sadek Nature 523, 226-230 (2015); doi:10.1038/nature14582 In this Letter we omitted to include the accession number for our RNAseq data.

Time-lapse fluorescence microscopy has transformed our ability to visualise dynamic cellular processes in vivo. However, the beating heart poses a major challenge due to the constant motion as it beats. One way around this is to stop the heart and image in static, but this inherently disrupts its function. Alternatively, the heart must be imaged continuously, and a single phase identified retrospectively, but this exposes the heart to high doses of light, which in itself is damaging. This limits our understanding of key processes in cardiac development. To overcome these limitations, we have developed a heartbeat-synchronised selective plane illumination microscopy (SPIM) system that actively triggers fluorescence image acquisition at a precise phase of the cardiac cycle. The system utilises brightfield videos to track heartbeat phase in real-time, computationally ‘freezing’ cardiac motion and capturing stable 3D image stacks across developmental time points without resorting to pharmacological arrest or high-intensity retrospective imaging. We applied this method to dual-labelled transgenic zebrafish lines in which cardiomyocyte nuclei and membranes are labelled, and combined it with automated cell tracking to reconstruct dynamic maps of cardiomyocyte behaviour between 3–5 days post-fertilisation. This approach enables quantitative, long-term, high-resolution imaging of the developing heart in vivo under physiological conditions.We are now extending this platform to zebrafish models of cardiomyopathy, specifically focusing on mutations of LMNA, to investigate early cellular mechanisms underlying disease, with the aim of uncovering new targets for early intervention in heart failure.

Hypertrophic cardiomyopathy (HCM) is a genetic disease often associated with sudden cardiac death and linked to Z-disc genetic variants. Alpha-actinin 2 (ACTN2) is a key Z-disc protein critical for stabilising the contractile muscle apparatus. A novel missense ACTN2 variant, M228T, was identified in 2014 in a family of 11 HCM patients. A previous in vivo study by our group showed embryonic lethality in mice with the M228T homozygous copy.1–3 However, the mechanism by which this missense variant impacts ACTN2 protein and leads to disease has not been widely investigated. In this study, we examine the functional implications of the ACTN2 M228T variant using biochemical and cellular models.The ACTN2 M228T variant was recombinantly expressed using E.coli, and the structural and thermal stability of the mutant protein was assessed. Functional implications of this variant were further assessed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).Structural modelling predictions revealed that the ACTN2 M228T variant adversely impacts the binding of ACTN2 to actin. Actin-binding assays showed increased binding affinity. Thermal assays showed decreased thermal stability, and enzymatic digestion using thermolysin showed reduced structural stability. Additional assays also showed increased aggregate formation.Moreover, the functional implications of M228T variant were assessed using an iPSC-CM model with mutant cardiomyocytes showing protein degradation, destabilisation, upregulation of hypertrophy and fibrosis. Mutant cells demonstrated impairment cellular quality control and autophagy, suggesting involvement of additional mechanisms.In summary, this study provides valuable insights into the impact of M228T variant on the protein structure and function. These approaches help facilitate our understanding of disease pathways in cardiomyopathy-linked ACTN2 variants.References Noureddine M, et al. Atrial electrical alterations with intact cardiac structure and contractile function in a mouse model of an HCM-linked ACTN2 variant. J Mol Cell Cardiol Plus. 2025 May 17;12:100455. doi: 10.1016/j.jmccpl.2025.100455. PMID: 40503000; PMCID: PMC12153375. Broadway-Stringer S, et al. Insights into the role of a cardiomyopathy-causing genetic variant in ACTN2. Cells. 2023 Feb 24;12(5):721. doi: 10.3390/cells12050721. PMID: 36899856; PMCID: PMC10001372. Girolami F, et al. Novel α-actinin 2 variant associated with familial hypertrophic cardiomyopathy and juvenile atrial arrhythmias: a massively parallel sequencing study. Circ Cardiovasc Genet. 2014 Dec;7(6):741–50. doi: 10.1161/CIRCGENETICS.113.000486. Epub 2014 Aug 30. PMID: 25173926.

Rhythmic contraction and relaxation of the myocardium impose continuous mechanical forces on cardiomyocyte nuclei via the cytoskeleton. Nuclear invaginations (NI), inward folds of the nuclear envelope, are observed in cardiomyocytes and are believed to be important for the cell contractile function.1 However, the mechanism behind the formation of NI and their functional roles remains unclear.Adult rat, control and 8 weeks, 16 weeks Heart Failure (HF) as well as mice SYNE+/+ and -/- cardiomyocytes were used in the study. Cells were subjected to pharmacological and mechanical stimulation. Super-resolution microscopy was used to visualize the nucleus and RNA labelling.Calcium handling was analysed via live confocal imaging with Fluo-4. Mechano-Scanning Ion Conductance Microscopy was obtained to measure cell Young’s modulus.Our findings show that NI are more frequent in left than right ventricular cardiomyocytes. Using pharmacological and genetic approaches, we identified actin and microtubule forces as key regulators of NI formation, highlighting the cytoskeleton’s role in their development. Additionally, the nucleoli were found to contribute to NI formation, suggesting an intranuclear pulling force mechanism. Functionally, NI loss decreased the perinuclear RNA and elevated nuclear Ca2+ levels. Lower NI density also increased DNA breaks and shifted H3K9me3 heterochromatin to H3K27me3. In HF cardiomyocytes, a progressive loss of NI was observed alongside nuclear morphological changes, with early nucleolar alterations preceding microtubular reorganization. Tissue analysis from patients with Dilated Cardiomyopathy revealed similar trends. Furthermore, biomechanical stress (high stiffness and pressure), significantly reduced NI formation.These novel findings suggest that NI are lost early in HF and are critical for maintaining nuclear function, structural integrity, and gene expression, making them essential in both, normal and pathological conditions.Reference Ljuobojevic S, Radulovic S, Leitinger G, et al. Early remodelling of perinuclear Ca2+ stores and nucleoplasmic Ca2+ signalling during the development of hypertrophy and heart failure. Circulation 2014;130:244–255.

RationaleMaladaptive cardiac hypertrophy predisposes individuals to heart failure. However, current treatments are inaccessible to drugs, and are therefore unsuitable for slowing the progression of hypertrophy. The cell-surface SLC proteins NHE (Slc9a1), NBCe1 (Slc4a4) and NBCn1 (Slc4a7), which are more therapeutically accessible, are critical in neutralising the pH of the highly metabolic cardiomyocytes, and are associated with both physiological and pathophysiological cellular growth. In particular, NBCn1, the electroneutral Na+-HCO3- transporter, is associated with pro-hypertrophic growth in cardiomyocytes and may be of therapeutic interest.MethodsCardiac hypertrophy was evoked in vivo in wildtype or Slc4a7 knockout mice by chemical stressors or abdominal aortic banding (AAB), and changes in cardiac function were assessed by echocardiography. Neonatal rat ventricular myocytes (NRVMs) treated with phenylephrine (PE). Assessment ofResultsNRVMs treated with 10μM PE at pH 7.4 and 24mM HCO3- exhibited increased GATA4 phosphorylation, Anp expression and SRB staining, indicative of increased protein production and consequent hypertrophy. However, these changes were attenuated both at pH 6.4 and in the presence of the pan-NBC inhibitor S0859. However, only Slc4a7 and not Slc4a4 expression was increased in hypertrophic NRVMs, and NBCn1 activity is higher in PE-treated NRVMs at pH 7.4, suggesting that HCO3- transport may be important. In fact, increased PE-induced GATA4 phosphorylation, Anp/Bnp expression and SRB staining was dependent on HCO3-. Moreover, HCO3- was critical for activation of the pro-hypertrophic mTOR signalling cascade and ribosome biogenesis.AAB increased diastolic LV diameter and wall thickness, associated with reduced ejection fraction and fractional shortening; however, these differences were ablated following treatment with S0859. Infusion of isoproterenol via minipumps also resulted in increased myocyte area, and increased Slc4a7 expression and NBCn1 activity in pro-hypertrophic hearts. Additionally, infusion of isoproterenol into Slc4a7 knockout mice exhibited reduced cardiac hypertrophy and left ventricular posterior wall thickness, associated with delayed progression of systolic dysfunction and better cardiac output and stroke volume, demonstrating the importance of the role of NBCn1 in vivo.ConclusionsNBCn1 is the predominant isoform involved in HCO3- dependent alkalinisation and cardiomyocyte hypertrophy, both in vitro and in vivo. It is therefore an attractive therapeutic target for the prevention of cardiac hypertrophy and heart failure.

The adult mammalian heart is the least regenerative organ in the body. Up to ~ a billion cardiomyocytes are lost after a heart attack. Recent advances to replace lost muscle (primary remuscularization) and recover cardiac function relied strongly on the relative accessibility of human embryonic stem cells (hESCs)-derived cardiomyocytes (CMs). To successfully regenerate the injured heart, hESCs-CMs must survive, proliferate, mature and integrate in vivo. However, the long-term behaviour of hESCs-CMs in vivo remains unpredictable, compounded by its pro-arrhythmic nature. Thus, we adapted a commonplace practice in drug development e.g. large-scale phenotypic drug screening, to systematically perturb each regulatory pathway in hESCs-CM to determine its composite behaviour in vitro in both injury and non-injury settings. We first developed a robust pipeline to enable small-scale phenotypic drug screening of hESCs-Cardiomyocytes with clinically applicable compounds such as beta-blockers, myosin inhibitors. Our outputs include fluorescence-labelled Ca2+ kinetics, subcellular live-cell imaging. However, we found that the hESC-CMs were highly variable with different maturation and contractility rates. To optimize the signal-to-noise ratio in our readouts, we aim to utilise the real-world data generated by our large-scale phenotypic drug screen to develop an in silico perturbation model of hESC-CMs. Ultimately, this data science-intensive approach would enable a better prediction of hESC-CMs behaviour in vitro and in vivo with wide-ranging applications in both cardiac regeneration and cardiovascular medicine specific to heart failure patients.

BackgroundUnderstanding cellular mechanisms driving heart failure (HF) progression requires characterisation of cardiomyocyte mechanics under varying loading conditions. While traditional isometric experiments offer limited insight into the dynamic cardiac function, work-loops models provide a more physiologically relevant framework for studying cardiac mechanics in-vitro. We employ a work-loop technique to examine myocyte mechanics under both physiological and pathological load, investigating the Frank-Starling relationship at a cellular level and the impact of high mechanical stress on cardiomyocyte function and structure.MethodsIsolated adult rat ventricular myocytes (n=15) were attached to a piezo-translator and force transducer (IonOptix, Milton, USA) and subjected to both isometric and workloop contractions at increasing sarcomere lengths (preload) at 1 Hz. Work-loops were configured with a preload of 10% and afterload of 50% of developed isometric force. Developed force and stroke work were averaged from ten stable traces. A high-preload protocol (paced at 3 Hz) was used to simulate volume overloaded conditions. Stroke work (area within the work-loop) was continuously monitored. Isometric force and cell morphology were measured pre- and post-experiment. Bathing solution was carefully replaced without disrupting cell position to assess for potential recovery.ResultsMyocytes exhibited exponential force augmentation with stretch, with a mean 3.45-fold (±0.58 SD) increase, but stretching beyond 2.2µM led to cell instability and death (figure 1A). Similarly, stroke work increased significantly from 1.7µm to 2.15µM, with a 5.42-fold (±0.96SD) rise from baseline (figure 1B). However, stable workloops could not be maintained at 2.2µM due to pro-arrhythmic behaviour or cell detachment from vigorous force-length changes. No optimal length plateau was demonstrated, unlike multicellular or in-vivo preparations. Under sustained high-preload conditions, significant mechanical deterioration was observed (30.3% force reduction. p=0.0012) within 8–10 minutes of stressed work-loops, accompanied by membrane blebbing and cellular distortion (figure 2). Buffer replacement or cell rest did not restore cellular function, indicating irreversible damage under pathologically high preload conditions.Abstract BS48 Figure 1(A) Developed Force of contraction as a function of sarcomeric length from 1.7 to 2.2µM (n=5 rats); (B) Stroke work from work-loops as a function of sarcomeric length with corresponding work-loops from 1.7 to 2.15µM sarcomeric lengths (n=5 rats)[Image Omitted. See PDF.]Abstract BS48 Figure 2(A) Series of high preload workloops at 3 Hz demonstrating a significant reduction in stroke work (area within work loop) over time; (B) Significant functional and structural impairment of the myocyte seen before and after the high preload mechanical stress (n=5 rats)[Image Omitted. See PDF.]ConclusionThis study shows that isolated cardiomyocytes, devoid of their natural extracellular matrix support, become vulnerable to irreversible damage when stretched beyond 2.2µM sarcomeric length. The mechanical vulnerability to brief periods of excessive preload supports the notion that sustained myocyte overdistension in volume-overloaded hearts can lead to irreversible cellular damage, potentially contributing to irreversible heart failure and replacement fibrosis. These results emphasize the importance of early intervention to reverse pathologically high stress to enhance the possibility of myocardial reverse remodelling and potential recovery.

IntroductionHypertrophic cardiomyopathy (HCM) is a common genetic disorder (~1 in 500 individuals) that leads to arrhythmias and heart failure. Despite advances in early diagnosis, treatment remains limited, with surgical myectomy as the primary intervention. Myofibroblasts drive myocardial fibrosis, disrupt cardiomyocyte electrophysiology, and increase arrhythmic risk. Previously, we demonstrated that myofibroblasts influence cardiomyocyte electrophysiology via paracrine signalling. Here, we investigate the role of myofibroblast-derived small extracellular vesicles (sEVs) in regulating cardiomyocyte electrophysiology and hypertrophy.MethodsPrimary cardiac fibroblasts (CFs) from HCM patient biopsy-derived explants were characterized for activation markers (αSMA, COL1A1, COL3A1, IL-11, IL-6, FAP, POSTN) using molecular and imaging techniques (qRT-PCR, flow cytometry, Western blotting, confocal microscopy, and collagen secretion assay). sEVs were isolated and characterized per MISEV 2023 guidelines. Their uptake in CFs and human iPSC-derived cardiomyocytes (hiPSC-CMs) was assessed by confocal microscopy. Functional effects of sEVs on hiPSC-CMs were evaluated via contractility assays, calcium transients (optical mapping), and action potential duration measurements. Molecular changes were analysed by qRT-PCR, RNA sequencing, and label-free quantitative proteomics (LC-MS/MS).ResultsHCM-derived CFs exhibited significantly increased activation markers compared to controls, with TGF-β1 receptor inhibition (SD208, 3 µM) reducing this activation. In contrast, TGF-β1 stimulation (5 ng/mL) significantly enhanced immortalized control CFs (ICFs) activation. sEVs from HCM myofibroblasts, ICFs (±TGF-β1), and HEK293 cells were enriched with EV markers (CD63, CD9, CD81) and lacked calnexin. TEM and NTA confirmed their morphology and size distribution. DiI-labelled sEVs were taken up by recipient CFs and hiPSC-CMs while no uptake of free dye control was observed.Functionally, myofibroblast-derived sEVs activated quiescent CFs, indicating their fibrosis promoting ability, and altered hiPSC-CM electrophysiology. Treated hiPSC-CMs exhibited increased spontaneous beating rates and metabolic activity, altered contractility, and disrupted calcium transients. Hypertrophic changes included increased cell size, nuclear count, and elevated expression of hypertrophy and calcium-handling genes (NPPA, NPPB, MYH6, RYR2, CACNA1C, ITP3R, PLN) suggested by qRT-PCR and RNA-sequencing. Proteomic analysis revealed enrichment of fibrosis-associated proteins, particularly Serpin E1/E2, in HCM myofibroblasts, their sEVs and sEV-treated hiPSC-CMs, indicating their role in driving hypertrophy.ConclusionsMyofibroblast-derived sEVs act as key mediators of intercellular signalling in HCM, driving cardiomyocyte hypertrophy and electrophysiological remodelling. These findings highlight their potential as novel therapeutic targets for less invasive interventions in cardiovascular disease.Abstract BS39 Figure 1Volcano plots generated using proteomic analysis to compare abundance of genes 2-fold upregulated and downregulated in (A) HCM myofibroblasts with and without SD208 treatment. (B) hiPSC-CM with and without sEV treatment. Several pro- fibrosis and hypertrophic proteins were enriched in HCM myofibroblasts and their sEVs relative to SD208 controls and in hiPSC-CMs treated with HCM sEVs relative to untreated cells[Figure omitted. See PDF]