UNASSIGNED: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease; DDX5 (dead-box helicase 5) is a master regulator of many RNA processes, although its function in heart physiology remains unclear.
UNASSIGNED: We assessed DDX5 expression in human failing hearts and a mouse HF model. To study the function of DDX5 in heart, we engineered cardiomyocyte-specific Ddx5 knockout mice. We overexpressed DDX5 in cardiomyocytes using adeno-associated virus serotype 9 and performed transverse aortic constriction to establish the murine HF model. The mechanisms underlined were subsequently investigated using immunoprecipitation-mass spectrometry, RNA-sequencing, alternative splicing analysis, and RNA immunoprecipitation sequencing.
UNASSIGNED: We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. DDX5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. We found that DDX5 regulated the aberrant splicing of Ca2+/calmodulin-dependent protein kinase IIδ (CamkIIδ), thus preventing the production of CaMKIIδA, which phosphorylates L-type calcium channel by serine residues of Cacna1c, leading to impaired Ca2+ homeostasis. In line with this, we found increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5-depleted cardiomyocytes. Using adeno-associated virus serotype 9 knockdown of CaMKIIδA partially rescued the cardiac dysfunction and HF in Ddx5 knockout mice.
UNASSIGNED: These findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes, identifying the DDX5 as a potential target for therapeutic intervention in HF.

UNASSIGNED: Obstructive sleep apnea (OSA) has been linked to various pathologies, including arrhythmias such as atrial fibrillation. Specific treatment options for OSA are mainly limited to symptomatic approaches. We previously showed that increased production of reactive oxygen species (ROS) stimulates late sodium current through the voltage-dependent Na+ channels via Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ), thereby increasing the propensity for arrhythmias. However, the impact on atrial intracellular Na+ homeostasis has never been demonstrated. Moreover, the patients often exhibit a broad range of comorbidities, making it difficult to ascertain the effects of OSA alone.
UNASSIGNED: We analyzed the effects of OSA on ROS production, cytosolic Na+ level, and rate of spontaneous arrhythmia in atrial cardiomyocytes isolated from an OSA mouse model free from comorbidities.
UNASSIGNED: OSA was induced in C57BL/6 wild-type and CaMKIIδ-knockout mice by polytetrafluorethylene (PTFE) injection into the tongue. After 8 weeks, their atrial cardiomyocytes were analyzed for cytosolic and mitochondrial ROS production via laser-scanning confocal microscopy. Quantifications of the cytosolic Na+ concentration and arrhythmia were performed by epifluorescence microscopy.
UNASSIGNED: PTFE treatment resulted in increased cytosolic and mitochondrial ROS production. Importantly, the cytosolic Na+ concentration was dramatically increased at various stimulation frequencies in the PTFE-treated mice, while the CaMKIIδ-knockout mice were protected. Accordingly, the rate of spontaneous Ca2+ release events increased in the wild-type PTFE mice while being impeded in the CaMKIIδ-knockout mice.
UNASSIGNED: Atrial Na+ concentration and propensity for spontaneous Ca2+ release events were higher in an OSA mouse model in a CaMKIIδ-dependent manner, which could have therapeutic implications.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.

Prolonged stimulation of β-adrenergic receptor (β-AR) can lead to sympathetic overactivity that causes pathologic cardiac hypertrophy and fibrosis, ultimately resulting in heart failure. Recent studies suggest that abnormal protein ubiquitylation may contribute to the pathogenesis of cardiac hypertrophy and remodeling. In this study, we demonstrated that deficiency of a deubiquitinase, Josephin domain-containing protein 2 (JOSD2), ameliorated isoprenaline (ISO)- and myocardial infarction (MI)-induced cardiac hypertrophy, fibrosis, and dysfunction both in vitro and in vivo. Conversely, JOSD2 overexpression aggravated ISO-induced cardiac pathology. Through comprehensive mass spectrometry analysis, we identified that JOSD2 interacts with Calcium-calmodulin-dependent protein kinase II (CaMKIIδ). JOSD2 directly hydrolyzes the K63-linked polyubiquitin chains on CaMKIIδ, thereby increasing the phosphorylation of CaMKIIδ and resulting in calcium mishandling, hypertrophy, and fibrosis in cardiomyocytes. In vivo experiments showed that the cardiac remodeling induced by JOSD2 overexpression could be reversed by the CaMKIIδ inhibitor KN-93. In conclusion, our study highlights the role of JOSD2 in mediating ISO-induced cardiac remodeling through the regulation of CaMKIIδ ubiquitination, and suggests its potential as a therapeutic target for combating the disease. Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary. All have been checked.

Obesity increases the risk of arterial hypertension in young adults and favors an early-onset cardiomyopathy by generating oxidative stress. In this sense, indiscriminate consumption of sucrose and fructose sweetened beverages from early ages causes obesity, however its consequences on the heart when there is a genetic predisposition to develop hypertension are not clear. We compared the effects of sucrose, fructose, and their combination in weanling male spontaneously hypertensive rats to determine the relationship between genetic hypertension, obesity, and consumption of these sugars on the degree of cardiac hypertrophy, oxidative stress and Ca2+/calmodulin dependent protein kinase II delta oxidation. Histological, biochemical, and Western blot studies were performed 12 weeks after treatment initiation. We found that chronic consumption of sucrose or fructose leads to obesity, exacerbates genetic arterial hypertension-induced metabolic alterations, and increases cardiac oxidative stress, Ca2+/calmodulin dependent protein kinase II delta oxidation and cardiac hypertrophy. Nonetheless, when sucrose and fructose are consumed together, metabolic alterations worsen and are accompanied by dilated cardiomyopathy. These data suggest that sucrose and fructose combined consumption starting from maternal weaning in rats with genetic predisposition to arterial hypertension accelerates the progression of cardiomyopathy resulting in an early dilated cardiomyopathy.

Necroptosis is a newly discovered mechanism of cell death, and its key regulatory role is attributed to the interaction of receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3. Ca2+/calmodulin-dependent protein kinase (CaMKII) is a newly discovered RIPK3 substrate, and its alternative splicing plays a fundamental role in cardiovascular diseases. In the present study, we aimed to explore the role and mechanism of necroptosis and alternative splicing of CaMKIIδ in myocardial hypertrophy. Transverse aortic constriction (TAC) was performed on wild-type and knockout mice to establish the model of myocardial hypertrophy. After 3 weeks, echocardiography, cardiac index, cross-sectional area of myocardial cells, hypertrophic gene expression, myocardial damage, and fibers were assessed. Moreover, we detected the levels of inflammatory factors (IL-6 and TNF-α) and examined the expressions of necroptosis-related proteins RIPK3, RIPK1, and phosphorylated MLKL. Meanwhile, we tested the expression levels of splicing factors ASF/SF2 and SC-35 in an attempt to explore CaMKII δ. The relationship between variable splicing disorder and the expression levels of splicing factors ASF/SF2 and SC-35. Further, we also investigated CaMKII activation, oxidative stress, and mitochondrial ultrastructure. In addition, wild-type mice were administered with a recombinant adeno-associated virus (AAV) carrying RIPK3, followed by TAC surgery to construct a model of myocardial hypertrophy, and the above-mentioned indicators were tested after 3 weeks. The results showed that RIPK3 deficiency could alleviate cardiac dysfunction, myocardial injury, aggravation of necrosis, and CaMKII activation induced by TAC surgery in mice with myocardial hypertrophy. Tail vein injection of AAV could reverse cardiac dysfunction, myocardial damage, aggravation of necrosis, and CaMKII activation in mice with myocardial hypertrophy. These results proved that RIPK3 could be used as a molecular intervention target for the prevention and treatment of myocardial hypertrophy.

BACKGROUND: Cyclosporine A (CsA)-induced cardiac interstitial fibrosis and cardiac hypertrophy are highly known phenomena; however, the basic mechanisms of CsA cardiotoxicity are unclear. The present study evaluated the role of the Transforming growth factor-beta (TGF-β)/Smad3/miR-29b signaling pathway and CaMKIIδ isoforms gene expression in cardiac remodeling under CsA exposure alone or combined with moderate exercise.
METHODS: A total of 24 male Wistar rats were divided into control, cyclosporine (30 mg/kg BW), and cyclosporine-exercise groups.
RESULTS: After 42 days of treatment, the findings revealed a significant decline in miR-29 and miR-30b-5p gene expression and an increase in gene expression of Smad3, calcium/calmodulin-dependent protein kinaseIIδ (CaMKIIδ) isoforms, Matrix Metalloproteinases (MMPs), protein expression of TGF-β, heart tissue protein carbonyl and oxidized LDL (Ox-LDL), and plasma LDL and cholesterol levels in the CsA-treated group compared to the control group. The CsA group presented greater histological heart changes such as fibrosis, necrosis, hemorrhage, infiltrated leukocyte, and left ventricular weight/heart weight than the control group. Moreover, combined moderate exercise and CsA relatively improved gene expression changes and histological alternations compared to the CsA group.
CONCLUSIONS: TGF-β-Smad3-miR-29 and CaMKIIδ isoforms may mainly contribute to the progression of heart fibrosis and hypertrophy due to CsA exposure, providing new insight into the pathogenesis and treatment of CsA-induced side effects on the heart tissue.

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca2+-sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM\'s Ca2+-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Cav1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.

Atherosclerosis (AS) is a chronic progressive inflammatory disease involving many cells. miR-145-5p mediates the biological phenotypes of human aortic vascular smooth muscle cells (HAVSMCs) and influences the progression of AS, but the potential mechanism needs further study.
Total RNA was extracted from patient plasma and arteries to determine the expression of miR-145-5p. The CaMKIIδ pathway and genes were predicted as the target of miR-145-5p by bioinformatics approaches. The interaction between miR-145-5p and CaMKIIδ was confirmed by RT-qPCR and Dual Luciferase Reporter Assay System. Western blot analysis, immunofluorescence staining, transmission electron microscopy (TEM) and protein tracing on HAVSMCs transduced with mCherry-GFP-LC3 lentiviral vectors to determine the mechanism by which miR-145-5p affects the atherosclerotic disease process.
The expression of miR-145-5p was downregulated in blood and arteries specimens of patients with coronary stenosis. Correspondingly, CaMKIIδ was upregulated and miR-145-5p was downregulated in hypoxic HAVSMCs. CaMKIIδ was predicted and confirmed as a downstream target of miR-145-5p. In addition, CaMKIIδ induced the upregulation of autophagy-related proteins by activating the AMPK/mTOR/ULK1 signalling pathway. Moreover, we confirmed that miR-145-5p inhibits CaMKIIδ expression by binding to a specific sequence in the CaMKIIδ 3\' UTR and affects autophagy. Crucially, CaMKIIδ was promoted by the downregulation of miR-145-5p and then activating autophagy in HAVSMCs through the AMPK/mTOR/ULK1 signalling pathway to affect the AS progress.
miR-145-5p regulates CaMKIIδ, leading to altered autophagy in HAVSMCs. This alteration plays an important role in AS progression.

Myocardial ischemia/reperfusion (I/R) injury poses a significant threat to human health. High level of reactive oxygen species (ROS) and calcium overload are the foremost causes of myocardial damage in I/R. Sulforaphane (SFN) is known for its promising antioxidant effect. Whether or not SFN has myocardial protective effect against I/R is largely unknown. This study aimed to investigate if SFN can protect myocardium from I/R injury. We found that mice or cells pre-treated with SFN showed improved cardiac functions and cell survival. SFN treatment inhibited the production of inflammatory cytokines and the increase of intracellular calcium induced by hypoxia-reperfusion (H/R), while mitochondria membrane potential was effectively maintained. Transcriptome analysis showed that CaMKIIδ expression was down-regulated by SFN treatment in I/R myocardium, while CaMKIIN2, the inhibitor of CaMKII, was upregulated. Knockdown of CaMKIIN2 not only led to increased level of total CaMKIIδ and the phosphorylated CaMKIIδ but also blocked the pro-survival effect of SFN for H/R cells. Moreover, CaMKIIN2 overexpression was sufficient to suppress CaMKIIδ activation and improve cell survival under H/R. Taken together, this study demonstrated that SFN exerts cardioprotective effect toward I/R injury through upregulating CaMKIIN2 and down-regulating CaMKIIδ.