Cardiomyocyte Function Research Articles

Structural color heart-on-a-chip for evaluating the myocardial protection effects of traditional Chinese medicine

Many Chinese medicines have shown protective effects on the heart, while their specific effects and modes of action are still lacking of intuitive studies. In this paper, we propose a structural color heart-on-a-chip from layer assembly for evaluating the myocardial protection effects of traditional Chinese medicine. The structural color scaffolds are imparted with a striped microstructure on one side for inducing the alignment of cardiomyocytes, and an inverse opal nanostructure on the other side for optical sensing. Because the scaffold could generate obvious color changes upon cardiomyocyte contraction, its integration into a microfluidic chip provides a simple screening platform to study the pre-protective effects of Chinese medicine against cardiac injury. We have demonstrated that with the induction of doxorubicin, the injured cardiomyocytes presented abnormal morphology, disappeared sarcomere and depressed beating behaviors that could be reflected by reduced structural color changes. On the contrary, traditional Chinese medicine pre-protection can increase the vitality of cardiomyocytes, reduce reactive oxygen species production, and improve the contractile function of cardiomyocytes, which is highly consistent with the known conclusion. These results indicated that our structural color heart-on-a-chip with real-time visualization characteristics could provide an ideal platform for traditional Chinese medicine development and drug screening.

Metabolic reprogramming via mitochondrial delivery for enhanced maturation of chemically induced cardiomyocyte-like cells.

Heart degenerative diseases pose a significant challenge due to the limited ability of native heart to restore lost cardiomyocytes. Direct cellular reprogramming technology, particularly the use of small molecules, has emerged as a promising solution to prepare functional cardiomyocyte through faster and safer processes without genetic modification. However, current methods of direct reprogramming often exhibit low conversion efficiencies and immature characteristics of the generated cardiomyocytes, limiting their use in regenerative medicine. This study proposes the use of mitochondrial delivery to metabolically reprogram chemically induced cardiomyocyte-like cells (CiCMs), fostering enhanced maturity and functionality. Our findings show that mitochondria sourced from high-energy-demand organs (liver, brain, and heart) can enhance structural maturation and metabolic functions. Notably, heart-derived mitochondria resulted in CiCMs with a higher oxygen consumption rate capacity, enhanced electrical functionality, and higher sensitivity to hypoxic condition. These results are related to metabolic changes caused by increased number and size of mitochondria and activated mitochondrial fusion after mitochondrial treatment. In conclusion, our study suggests that mitochondrial delivery into CiCMs can be an effective strategy to promote cellular maturation, potentially contributing to the advancement of regenerative medicine and disease modeling.

Suppression of Dad1 induces cardiomyocyte death by weakening cell adhesion.

As cardiomyocyte loss causes heart failure, inhibition of cardiomyocyte death may be a therapeutic strategy against heart failure. In this study, we have identified Defender against cell death 1 (Dad1) as a candidate regulator of cardiomyocyte death, using complementary DNA microarray and siRNA knockdown screening. Dad1 is a subunit of oligosaccharyltransferase (OST) complex that is responsible for protein N-glycosylation; however, its function in cardiomyocytes remains unknown. Importantly, the knockdown of Dad1 using siRNA reduced the viability of neonatal rat cardiomyocytes (NRCMs), accompanied by cleaved caspase3 expression, independent of endoplasmic reticulum stress. Dad1 knockdown impaired cell spreading and reduced myofibrillogenesis in NRCMs, suggesting that Dad1 knockdown induced anoikis, apoptosis by disrupting cell-matrix interactions. Consistently, knockdown of Dad1 impaired N-glycosylation of integrins α5 and β1, accompanied by inactivation of focal adhesion kinase. When cell adhesion was enhanced using adhesamine, fibronectin, or collagen type IV, cardiomyocyte death induced by Dad1 knockdown was reduced. Dad1 knockdown decreased the expression of staurosporine and temperature sensitive 3A (Stt3A), a catalytic subunit of OST complex. Interestingly, Stt3A knockdown using Stt3A siRNA reduced the expression of Dad1, indicating that both Dad1 and Stt3A were required for OST stabilization. In conclusion, Dad1 plays an important role in maintaining the expression of mature N-glycosylated integrins and their downstream signaling molecules to suppress cardiomyocyte anoikis.

The role of protein O-GlcNAcylation in diabetic cardiomyopathy.

It is well established that diabetes markedly increases the risk of multiple types of heart disease including heart failure. However, despite substantial improvements in the treatment of heart failure in recent decades the relative increased risk associated with diabetes remains unchanged. There is increasing appreciation of the importance of the post translational modification by O-linked-N-acetylglucosamine (O-GlcNAc) of serine and threonine residues on proteins in regulating cardiomyocyte function and mediating stress responses. In response to diabetes there is a sustained increase in cardiac O-GlcNAc levels, which has been attributed to many of the adverse effects of diabetes on the heart. Here we provide an overview of potential mechanisms by which increased cardiac O-GlcNAcylation contributes to the adverse effects on the heart and highlight some of the key gaps in our knowledge.

New insights into FGF21 alleviates diabetic cardiomyopathy by suppressing ferroptosis: a commentary.

Diabetic cardiomyopathy (DCM) is a severe cardiovascular complication of diabetes characterized by myocardial hypertrophy, fibrosis, and impaired cardiac function. Fibroblast growth factor 21 (FGF21) has emerged as a promising therapeutic target due to its antifibrotic, antioxidant, and anti-inflammatory properties. Our commentary summarizes and affirms the recent study by Wang et al., which demonstrates the significant role of ferroptosis in DCM pathogenesis. FGF21 has shown promise as a therapeutic target for DCM, potentially inhibiting ferroptosis, mitigating oxidative damage, and protecting cardiomyocyte function. Mechanistically, the study identified ATF4 as an upstream regulator of FGF21 in DCM, revealing that FGF21 directly interacts with ferritin and extends its half-life, thus inhibiting ferroptosis in DCM. These findings provide a theoretical basis for understanding the pathogenesis and treatment of DCM. Our commentary suggests that future studies should explore the role of non-cardiomyocyte cell types in DCM, verify findings with clinical samples, and address comprehensive methods for ferroptosis detection. Additionally, we discuss the clinical application and future potential of FGF21-based therapies for DCM. Such efforts may contribute to advancing DCM diagnosis and treatment, fostering the development of innovative therapeutic strategies.

Lysophosphatidylethanolamine improves diastolic dysfunction by alleviating mitochondrial injury in the aging heart

Diastolic dysfunction in aging mice is linked to mitochondrial abnormalities, including mitochondrial morphology disorders and decreases in membrane potential. Studies also show that aberrant mitochondrial lipid metabolism impairs mitochondrial function in aging cardiomyocytes. Our lipidomic analysis revealed that phosphatidylethanolamine (PE) levels were significantly decreased in aging myocardial mitochondria. Here, we investigated whether reduction in PE levels in myocardial mitochondria contributes to mitochondrial injury as well as HFpEF pathogenesis, and whether modulation of PE levels could ameliorate aging-induced HFpEF. Echocardiography was used to assess cardiac diastolic function in adult and aging mice treated with lysophosphatidylethanolamine (LPE) or saline. Mitochondrial morphologies from tissue samples were evaluated by transmission electron microscopy (TEM), while mitochondrial membrane potential and reactive oxygen species (ROS) levels were assessed using JC-1, MitoSOX and DCFH-DA detection assays. We performed GO enrichment analysis between adult and aging mice and discovered significant enrichment in transcriptional programs associated with mitochondria and lipid metabolism. Also, mitochondrial PE levels were significantly decreased in aging cardiomyocytes. Treatment with LPE significantly enhanced PE content in aging mice and improved the structure of mitochondria in cardiac cells. Also, LPE treatment protected against aging-induced deterioration of mitochondrial injury, as evidenced by increased mitochondrial membrane potential and decreased mitochondrial ROS. Furthermore, treatment with LPE alleviated severe diastolic dysfunction in aging mice. Taken together, our results suggest that LPE treatment enhances PE levels in mitochondria and ameliorates aging-induced diastolic dysfunction in mice through a mechanism involving improved mitochondrial structure and function.

Maladaptive cardiomyocyte calcium handling in adult offspring of hypoxic pregnancy: protection by antenatal maternal melatonin.

Chronic fetal hypoxia is one of the most common complications of pregnancy and can programme cardiac abnormalities in adult offspring including ventricular remodelling, diastolic dysfunction and sympathetic dominance. However, the underlying mechanisms at the level of the cardiomyocyte are unknown, preventing the identification of targets for therapeutic intervention. Therefore, we aimed to link echocardiographic data with cardiomyocyte function to reveal cellular mechanism for cardiac dysfunction in rat offspring from hypoxic pregnancy. Further, we investigated the potential of maternal treatment with melatonin as antenatal antioxidant therapy. Wistar rats were randomly allocated into normoxic (21% O2) or hypoxic (13% O2) pregnancy with or without melatonin treatment (5µg/ml; normoxic melatonin in the maternal drinking water from gestational day 6 to 20 (term=22days). After delivery, male and female offspring were maintained to adulthood (16weeks). Cardiomyocytes were isolated from the left and right ventricles, and calcium (Ca2+) handling was investigated in field-stimulated myocytes. Systolic and diastolic function was negatively impacted in male and female offspring of hypoxic pregnancy demonstrating biventricular systolic and diastolic dysfunction and compensatory increases in cardiac output. Ca2+ transients from isolated cardiomyocytes in offspring of both sexes in hypoxic pregnancy displayed diastolic dysfunction with a reduced rate of [Ca2+]i recovery. Cardiac and cardiomyocyte dysfunction in male and female adult offspring was ameliorated by maternal antenatal treatment with melatonin in hypoxic pregnancy. Therefore, cardiomyocyte Ca2+ mishandling provides a cellular mechanism explaining functional deficits in hearts of male and female offspring in pregnancies complicated by chronic fetal hypoxia. KEY POINTS: This study identified significant changes in Ca2+ handling within cardiomyocytes isolated from offspring of hypoxic pregnancy including reduced systolic Ca2+ transients, impaired diastolic recovery of [Ca2+]i and a greater increase in systolic [Ca2+]i amplitude to β-adrenergic stimulation. These changes in cardiomyocyte Ca2+ handling help to explain dysregulation of biventricular systolic and diastolic dysfunction determined by echocardiography. The data show protection against maladaptive cardiomyocyte calcium handling and thereby improvement in cardiac function in adult offspring of hypoxic pregnancy treated with melatonin with doses lower than those recommended for overcoming jet lag in humans. Melatonin treatment alone in healthy pregnancy did cause some alterations in cardiac structure. Therefore, maternal treatment with melatonin should only be given to pregnancies affected by chronic fetal hypoxia.

SIRT1-dependent regulation of mitochondrial metabolism participates in miR-30a-5p-mediated cardiac remodeling post-myocardial infarction

Myocardial infarction-triggered myocardial remodeling is fatal for therapies. The miR-30 family is an essential component of several physiological and pathological processes. Previous studies have proved that the miR-30 family may contribute to regulating myocardial infarction. This study aimed to demonstrate that the combination of miR-30a-5p and mitochondrial metabolism recapitulates the critical features for remodeling post-myocardial infarction. Using gain- and loss-of-function of miR-30a-5p in mice, we found miR-30a-5p is highly expressed in the heart and is reduced in infarcted hearts. Further evidence showed that miR-30a-5p acts as a protective molecule to maintain myocardial remodeling, fibrosis, and mitochondrial structure. Mitochondrial function, ATP production, and mitochondrial respiratory chain proteins were positively regulated by miR-30a-5p. Mechanistically, alterations in these properties depend on SIRT1, which modulates miR-30a-5p-regulated mitochondrial metabolism. Remarkably, reactivation of SIRT1 prevented miR-30a-5p deficiency-aggravated myocardial infarction-induced myocardial remodeling. These data identified miR-30a-5p as a critical modulator of mitochondrial function in cardiomyocytes and revealed that the miR-30a-5p-SIRT1-mitochondria network is essential for myocardial infarction-induced cardiac remodeling.

Abstract 4122257: Inhibition of Neutrophil Extracellular Traps Prevents Heart Failure by Preserving Mitochondrial Function of Cardiomyocytes in Mice

Background: We have previously demonstrated that neutrophil extracellular traps (NETs) in myocardial tissue are associated with cardiac dysfunction and adverse outcomes in patients with heart failure with reduced ejection fraction. Here, we aimed to elucidate the underlying mechanisms and to clarify whether inhibiting NETs could rescue the cardiac phenotype using ex vivo and in vivo models. Methods and Results: Given that peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs, PAD4 knockout mice were used for this study. First, we assessed the mitochondrial function of cardiomyocytes by conducting real-time measurements of the oxygen consumption rate in ex vivo . Extracellular flux analysis revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of adult mouse cardiomyocytes, while these effects were abolished when PAD4 in neutrophils was genetically ablated (Figure 1). In a murine model of pressure overload-induced heart failure, transverse aortic constriction (TAC) operation was performed on wild-type and PAD4 knockout mice. Immunohistological analysis demonstrated that TAC induced the formation of NETs in the myocardium, with the greatest number of NETs occurring at 1 day, persisting into the failing stage at 4 weeks in wild-type mice, while PAD4-deficient hearts displayed no presence of NETs in the heart tissue (Figure 2). Four weeks after TAC, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4 knockout mice displayed preserved left ventricular ejection fraction (Figure 2). Finally, we investigated the bioenergetics of cardiomyocytes isolated from PAD4 knockout mice after TAC. Maximal mitochondrial oxygen consumption rate in cardiomyocytes isolated from TAC-operated PAD4 knockout mice was significantly higher than in those from TAC-operated wild-type mice (Figure 3). Conclusion: Our data suggest that NETs directly derived impairment of mitochondrial function in cardiomyocytes. Inhibiting NETs represents a potential therapeutic approach for heart failure, preventing mitochondrial dysfunction of cardiomyocytes.

Abstract 4126304: A Cardiac Targeting Peptide Linked to miRNA106a Targets and Suppresses Genes Known to Cause Heart Failure: Reversing Heart Failure at the Source

Background: About one in five adults at age 40 will develop heart failure (HF) during their lifetime. Risk factors for HF (e.g., hypertension, etc.) alter cardiomyocytes structure and function resulting in HF. At the cellular level, these changes consist of mis/over-expression of genes that regulate cardiac identity (e.g., CamK2d, PKC, etc). The current paradigm for treating HF is pharmacological intervention or surgery; however, with few exceptions, the condition worsens with time. We are proposing a paradigm-shift for treating HF from a drug-centric system to a molecular approach that would reverse hypertrophy and adverse remodeling. Methods: A cardiac targeting peptide (CTP) was generated and linked by disulfide bond to miRNA106a, which was then introduced into a HF mouse model to measure its ability to reverse multiple heart failure parameters. CTP-miRNA106a was also introduced into a human cardiac cell line, followed by multiple analyses including Western Blot, qPCR, FACS, immunofluorescence all used to identify mechanism(s) utilized by CTP-miRNA106a to reverse HF characteristics. Results: Bio-distribution studies showed CTP delivered its miRNA106a cargo specifically to the heart within 30 mins, followed by clearance of CTP from the heart to the kidneys and liver by 3-5hrs post systemic drug injection. MiRNA106a persisted in cardiomyocytes until day 7 (latest tested time-point). CTP-miRNA106a reversed Ang2/Iso induced hypertrophy in 90% of the experimental mice ( Fig1 ). We also identified two potential HF intracellular signaling mechanisms (PLCb1/PKC/IP3 and NfkB) targeted by CTP-miRNA106a that could benefit both types of HF, HFrEF and HFpEF. PLCβ1 is a direct target for miRNA106a while the Nfkb pathway is indirectly targeted by decreased Camk2d (an miRNA106a target) signaling to IκBα. NfkB activity is quelled by miRNA106a. Conclusions: Our approach utilizes the function of miRNA106a to downregulate genes involved in cardiac hypertrophy and inflammation through the PLCb1 and CamKIId/Nfkb pathways. Most importantly, using a CTP-miRNA106a, we show delivery of miRNA106a cargo is specific to cardiomyocytes both in vitro and in vivo and that once delivered, many HF parameters including hypertrophy are reversed.

Abstract 4139187: Electrophysiological and Mechanical Characterization of Human Ventricular Myocardium from Patients with Primary or Secondary Cardiac Hypertrophy

Left-ventricular hypertrophy (LVH) is an adaptive condition to hemodynamic stress often involving the left ventricle (LV). This pathological condition is associated with clinical complications such as ventricular arrhythmias and diastolic dysfunction, the molecular and cellular mechanisms of which have been poorly investigated in the human heart. We collected myocardial samples from the upper interventricular septum of 132 patients with hypertrophic cardiomyopathy (HCM), 42 patients with aortic stenosis and severe LVH (AoS-LVH) and 12 non-failing non-hypertrophic patients with valve disease (NF-NH), who underwent myectomy operations at our cardiac surgery center. Samples were used to isolate single viable cardiomyocytes from the left ventricle to perform patch-clamp electrophysiological studies and intracellular calcium measurements with fluorescent dyes. Intact trabeculae were also dissected to perform isometric force measurements of electrically-stimulated twitches. Single-cell patch-clamp studies revealed a marked prolongation of action potential duration (APD) in HCM (N=82, mean APD at 90% repolarization=763±252ms at 0.5Hz) and AoS-LVH (N=22, APD90%=579±147ms) samples with respect to NF-NH (N=12, APD90%=447±88ms). In both HCM and AoS-LVH cardiomyocytes, APD prolongation was associated with increased late-Na + current with respect to NF-NH, while L-type Ca 2+ current was enlarged only in HCM samples. Ca 2+ -fluorescence studies revealed markedly slower Ca-transient (CaT) kinetics in myocardial samples from HCM (N=38, mean CaT 50% decay time at 0.5 Hz = 658±179ms) and AoS-LVH patients (N=9, CaT50%= 568±187ms), when compared with NF-NH samples (N=8, CaT50%=283±59ms), paralleled by elevated diastolic [Ca 2+ ]. Twitch force measurements in intact trabeculae revealed prolonged isometric contractions in HCM (N=78, overall twitch duration at 0.5Hz= 730±147ms) and in AoS-LVH patient-samples (N=24, TwD=669±116ms), as compared with NF-NH (N=7, TwD=511±73ms). Force-frequency relationship was flat in pathological samples, while NF-NH trabeculae showed a clear increase in twitch amplitude while increasing pacing rate to 2Hz. Our results suggest that the main features of functional cardiomyocyte remodeling (that is, changes in the expression and/or function of ion-channel and EC-coupling proteins) are qualitatively similar in primary vs. secondary LVH, albeit abnormalities are quantitatively more extensive in HCM samples.

MicroRNA-24 therapeutic potentials in infarction, stroke, and diabetic complications.

The prevalence of cardiovascular events, stroke, and diabetes worldwide underscores the urgent need for effective and minimally invasive treatments. With nearly 20million annual casualties attributed to cardiovascular diseases and an estimated 463million people living with diabetes in 2022. Identifying promising therapeutic candidates is paramount. MicroRNAs, short nucleic acids involved in regulating gene expression, emerge as potential game-changers. Among these, microRNA-24 (miR-24), a hypoxia-sensitive player in endothelial vessels, has protective roles against diverse vascular complications. Following heart infarction and stroke, elevating miR-24 expression proves beneficial by mitigating oxidative stress, inflammation, and apoptosis while enhancing cell survival. It reduces cardiac fibrosis in heart disease, regulates aberrant angiogenesis in cerebral hemorrhagic strokes, and enhances the functionality of cardiomyocytes and brain neurons. In diabetic conditions, augmenting miR-24 expression mitigates complications. Further, being miR-24 also expressed by the skeletal muscle (i.e., myo-miR) in response to exercise, this miRNA may participate in the complex molecular network that systemically spreads the beneficial effects of physical exercise. This review provides a comprehensive vision of the molecular mechanisms underpinning the miR-24 protective effects, offering new insights into its therapeutic potential and proposing a novel avenue for medical intervention.

Klf9 is essential for cardiac mitochondrial homeostasis.

Mitochondrial dynamics and mitophagy are intimately linked physiological processes that are essential for cardiac homeostasis. Here we show that cardiac Krüppel-like factor 9 (Klf9) is dysregulated in human and rodent cardiomyopathy. Both global and cardiac-specific Klf9-deficient mice displayed hypertrophic cardiomyopathy. Klf9 knockout led to mitochondrial disarray and fragmentation, impairing mitochondrial respiratory function in cardiomyocytes. Furthermore, cardiac Klf9 deficiency inhibited mitophagy, thereby causing accumulation of dysfunctional mitochondria and acceleration of heart failure in response to angiotensin II treatment. In contrast, cardiac-specific Klf9 transgene improved cardiac systolic function. Mechanistically, Klf9 knockout decreased the expression of PGC-1α and its target genes involved in mitochondrial energy metabolism. Moreover, Klf9 controlled the expression of Mfn2, thereby regulating mitochondrial dynamics and mitophagy. Finally, adeno-associated virus-mediated Mfn2 rescue in Klf9-CKO hearts improved cardiac mitochondrial and systolic function. Thus, Klf9 integrates cardiac energy metabolism, mitochondrial dynamics and mitophagy. Modulating Klf9 activity may have therapeutic potential in the treatment of heart failure.

Induced pluripotent stem cell-derived cardiomyocyte in vitro models: benchmarking progress and ongoing challenges.

Recent innovations in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs) have unlocked a viable path to creating in vitro cardiac models. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) remain immature, leading many in the field to explore approaches to enhance cell and tissue maturation. Here, we systematically analyzed 300 studies using hiPSC-CM models to determine common fabrication, maturation and assessment techniques used to evaluate cardiomyocyte functionality and maturity and compiled the data into an open-access database. Based on this analysis, we present the diversity of, and current trends in, in vitro models and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology and gene expression and note field-wide improvements over time. Finally, we discuss opportunities to collectively pursue the shared goal of hiPSC-CM model development, maturation and assessment that we believe are critical for engineering mature cardiac tissue.

Septins regulate heart contractility through modulation of cardiomyocyte store-operated calcium entry.

Highly regulated cardiomyocyte Ca 2+ fluxes drive heart contractions. Recent findings from multiple organisms demonstrate that the specific Ca 2+ transport mechanism known as store-operated Ca 2+ entry (SOCE) is essential in cardiomyocytes for proper heart function, and SOCE dysregulation results in cardiomyopathy. Mechanisms that regulate SOCE in cardiomyocytes are poorly understood. Here we tested the role of cytoskeletal septin proteins in cardiomyocyte SOCE regulation. Septins are essential SOCE modulators in other cell types, but septin functions in cardiomyocytes are nearly completely unexplored. We show using targeted genetics and intravital imaging of heart contractility in Drosophila that cardiomyocyte-specific depletion of septins 1, 2, and 4 results in heart dilation that phenocopies the effects of SOCE suppression. Heart dilation caused by septin 2 depletion was suppressed by SOCE upregulation, supporting the hypothesis that septin 2 is required in cardiomyocytes for sufficient SOCE function. A major function of SOCE is to support SERCA-dependent sarco/endoplasmic reticulum (S/ER) Ca 2+ stores, and augmenting S/ER store filling by SERCA overexpression also suppressed the septin 2 phenotype. We also ruled out several potential SOCE-independent septin functions, as septin 2 phenotypes were not due to septin function during development and septin 2 was not required for z-disk organization as defined by α-actinin labeling. These results demonstrate, for the first time, an essential role of septins in cardiomyocyte physiology and heart function that is due, at least in part, to septin regulation of SOCE function.

Intracellular delivery of a phospholamban-targeting aptamer using cardiomyocyte-internalizing aptamers

The sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a)–phospholamban (PLN) system within the sarcoplasmic reticulum is crucial for regulating intracellular Ca2+ cycling in ventricular cardiomyocytes. Given that impaired Ca2+ cycling is associated with heart failure, modulating SERCA2a activity represents a promising therapeutic strategy. Previously, we engineered an RNA aptamer (Apt30) that binds to PLN, thereby activating SERCA2a by alleviating PLN's inhibitory effect. However, Apt30 alone cannot reach intracellular PLN, necessitating the development of a mechanism for its specific internalization into cardiomyocytes.Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we isolated RNA aptamers capable of internalizing into cardiomyocytes. These aptamers demonstrated sub-micromolar EC50 values for cardiomyocyte internalization and exhibited significantly reduced activity against various non-myocardial cells, highlighting their specificity for cardiomyocytes. Moreover, some of these cardiomyocyte-internalizing aptamers could be linked to Apt30 as a single RNA strand without compromising their internalization efficacy. Supplementing the culture medium with these hybrid aptamers enhanced Ca2+ transients and contractile function in rat cardiomyocytes. These findings provide critical insights for developing novel therapeutics directly acting on PLN in cardiomyocytes, potentially compensating for the disadvantages of conventional methods that involve viral vector-mediated intracellular transduction or alterations in endogenous protein expression.

Post-acute sequelae of SARS-CoV-2 cardiovascular symptoms are associated with trace-level cytokines that affect cardiomyocyte function

An estimated 65 million people globally suffer from post-acute sequelae of COVID-19 (PASC), with many experiencing cardiovascular symptoms (PASC-CVS) like chest pain and heart palpitations. This study examines the role of chronic inflammation in PASC-CVS, particularly in individuals with symptoms persisting over a year after infection. Blood samples from three groups—recovered individuals, those with prolonged PASC-CVS and SARS-CoV-2-negative individuals—revealed that those with PASC-CVS had a blood signature linked to inflammation. Trace-level pro-inflammatory cytokines were detected in the plasma from donors with PASC-CVS 18 months post infection using nanotechnology. Importantly, these trace-level cytokines affected the function of primary human cardiomyocytes. Plasma proteomics also demonstrated higher levels of complement and coagulation proteins in the plasma from patients with PASC-CVS. This study highlights chronic inflammation’s role in the symptoms of PASC-CVS.

SNORD3A: a new possible circulating biomarker of human heart failure

Abstract Background Heart failure (HF) is a complex clinical syndrome and the leading cause of mortality, morbidity and hospitalization in industrialized countries. Human cardiac biopsies are invaluable resources for identifying cardiac signaling pathways, however blood fractions and peripheral blood mononuclear cells (PBMCs) could be used as a source of surrogate biomarkers of signaling pathway activation in human heart failure. Purpose In the present study we aimed to identify novel circulating biomarkers mirroring human myocardial signaling in HF and to study the role played by SNORD3A in cardiomyocyte survival and death. Methods Cardiac left ventricle samples and PBLs from 8 HF patients undergoing heart transplantation and 2 control patients (CTRL) were analyzed by RNA sequencing (RNASeq) to identify transcripts that were regulated in both hearts and PBLs. Five identified transcripts, KCNQOT1, MIAT, SCRN1, MALAT1 and SNORD3A, were then validated by quantitative real-time PCR in PBMCs and in LVs. Next, we analyzed the expression of the Small Nucleolar RNA (snoRNA) SNORD3A in LVs and PBMCs from 8-week-old wild type C57BL/6 mice with pressure overload-induced HF by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. To further investigate the role of SNORD3A in cardiomyocyte function and survival, SNORD3A antisense oligonucleotides (SNOaso) and scramble control sequences (GFPaso) were synthesized and tested in cardiomyoblasts H9C2 cells under normoxic or hypoxic conditions to evaluate SNORD3A transcription levels, cell death and protein synthesis. Results SNORD3A expression was significantly increased in both LVs and PBMCs from HF patients compared to control subjects. Consistent with these results, SNORD3A levels were increased both in PBMCs and LVs from TAC mice compared to sham. In vitro hypoxia significantly increased SNORD3A expression levels in H9C2 cells, compared to normoxia. After SNOaso transfection, SNORD3A transcripts were downregulated, and they were further reduced following 3-hour-hypoxia. Depletion of SNORD3A levels increased cardiomyocyte death and reduced protein synthesis in H9C2 cells. Conclusions Our data suggest that SNORD3A might be involved in the regulation of cardiomyocyte survival in HF and could be useful for diagnostic and prognostic applications in HF.

Calcitonin signalling system regulates function and arhythmogenicity of atrial cardiomyocytes

Abstract Background Atrial fibrillation (AF) poses a significant therapeutic challenge due to myocardial remodelling, involving both structural and electrical alterations. Recent investigations have shed light on the role of atrial cardiomyocytes (CMs) in secreting Calcitonin (CT), a factor crucial for maintaining atrial tissue integrity. Dysregulated CT secretion in AF showed to contribute to fibrotic tissue production by atrial fibroblasts, exacerbating arrhythmogenesis [1]. However, the direct impact of CT on CM function and arrhythmogenicity remains unclear, driving the objectives of our study. Methods/Results Serum samples from 20 cardiac surgery patients revealed a noteworthy association between higher pre-operative CT levels and a significant ~2.8-fold reduction in the incidence of post-operative AF (poAF). Using freshly isolated atrial guinea pig CMs, confirmed (by qPCR and immunofluorescence) that CMs express CT-receptor (CTR) to enable CT actions in the cell. Functional studies found that CT administration inhibits spontaneous calcium (Ca2+)-release events induced by pacing and decreases the Ca2+ transients amplitude (at 2 Hz; IonOptix μstep system) in fura2-loaded CMs (n=22 cells) in a concentration-dependent manner. Atrial iPSC-CMs transduced with global RGECO sensor (Genetically encoded Ca2+ indicator) and treated with 15 pM CT showed a reduction in the beat rate when paced at 2Hz, and a significantly increased the time to 50% baseline. Evaluation of the expression and phosphorylation of selected Ca2+ handling proteins, which may potentially account for the observed changes in Ca2+ transients, showed no differences in total or phospho-(ser2808) ryanodine receptor, and SERCA2α in response to CT but an increase in phospho-(Ser16) Phospholamban. Conclusion Our findings highlight the effects of CT on atrial CM function. Maintaining physiological CT levels offer a promising approach for managing AF clinically, potentially through the use of already in clinical use CT-analogues.

Deletion of nephrocystin 4 exacerbates cardiac function and survival rate in a mouse model of cardio-renal syndrome

Abstract Background Cardiorenal syndrome (CRS), characterized by cardiac and renal dysfunction, is an increasing health problem associated with high mortality rate. Nephrocystin 4 (NPHP4) gene is the major cause of end-stage renal disease in children. We have reported NPHP4 single nucleotide polymorphism is the genetic risk for CRS and subsequent cardiovascular events in general population. However, the functional role of Nphp4 in the development of CRS has never been examined yet. Methods and Results We generated systemic Nphp4 knockout (KO) mice deleting exon 3 to 8 using CRISPR-Cas9 system. RNA sequence analysis of heart and kidney tissues demonstrated that gene-sets related to mitochondrial function were significantly downregulated in Nphp4 KO mice compared to their wild-type (WT) littermates. Nphp4 KO mice showed less cardiac hypertrophy, however, exacerbated cardiac function and survival compared to WT mice in a CRS model induced by thoracic transverse aortic constriction. Nphp4 located in endosome and cell membrane in H9C2 cardiomyocytes. Immunoprecipitation confirmed protein interaction between Nphp4 and HECT-type E3 ligase Itch. Furthermore, we found that Itch targeted Lats2 and insulin-like growth factor-1 receptor in cardiomyocytes. Knockdown of Nphp4 activated Hippo signaling and worsened mitochondrial function in H9C2 cardiomyocytes through the inhibition of Itch-dependent Lats2 degradation. Knockdown of Nphp4 inhibited insulin-like growth factor-1 signaling through the disturbance of Itch-mediated endosome function. Conclusions Deletion of Nphp4 impaired compensatory cardiac hypertrophy and exacerbated cardiac function and survival in a mouse CRS model through mitochondrial dysfunction. Nphp4 mediated Hippo signaling and insulin-like growth factor-1 signaling through the interaction with Itch. NPHP4 could be the novel therapeutic target in CRS.figure1figure2