Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

Protective effect of SERCA2a-SUMOylation by SUMO-1 on diabetes-induced atherosclerosis and aortic vascular injury.

Diabetes is a major risk factor for cardiovascular disease. However, the exact mechanism by which diabetes contributes to vascular damage is not fully understood. The aim of this study was to investigate the role of SUMO-1 mediated SERCA2a SUMOylation in the development of atherosclerotic vascular injury associated with diabetes mellitus. ApoE-/- mice were treated with streptozotocin (STZ) injection combined with high-fat feeding to simulate diabetic atherosclerosis and vascular injury. Human aortic vascular smooth muscle cells (HAVSMCs) were treated with high glucose (HG, 33.3 mM) and palmitic acid (PA, 200 µM) for 24 h to mimic a model of diabetes-induced vascular injury in vitro. Aortic vascular function, phenotypic conversion, migration, proliferation, intracellular Ca2+ concentration, the levels of small ubiquitin-like modifier type 1 (SUMO1), SERCA2a and SUMOylated SERCA2a were detected. Diabetes-induced atherosclerotic mice presented obvious atherosclerotic plaques and vascular injury, companied by significantly lower levels of SUMO1 and SERCA2a in aorta. HG and PA treatment in HAVSMCs reduced the expressions of SUMO1, SERCA2a and SUMOylated SERCA2a, facilitated the HAVSMCs phenotypic transformation, proliferation and migration, attenuated the Ca2+ transport, and increased the resting intracellular Ca2+ concentration. We also confirmed that SUMO1 directly bound to SERCA2a in HAVSMCs. Overexpression of SUMO1 restored the function and phenotypic contractile ability of HAVSMCs by upregulating SERCA2a SUMOylation, thereby alleviating HG and PA-induced vascular injury. These observations suggest an essential role of SUMO1 to protect diabetes-induced atherosclerosis and aortic vascular injury by the regulation of SERCA2a-SUMOylation and calcium homeostasis.

Qifu Yixin Formula Improves Heart Failure by Enhancing ß-Arrestin2 Mediated the SUMOylation of SERCA2a.

Purpose: This study aimed to elucidate the protective mechanism of Traditional Chinese Medicine (TCM) Qifu Yixin formula (QFYXF) to improve heart failure (HF) by promoting ß-arrestin2 (ß-arr2)-mediated SERCA2a SUMOylation. Materials and Methods: The transverse aortic constriction (TAC)-induced HF mice were treated with QFYXF or carvedilol for 8 weeks. ß-arr2-KO mice and their littermate wild-type (WT) mice were used as controls. Neonatal rat cardiomyocytes (NRCMs) were used in vitro. Cardiac function was evaluated by echocardiography and serum NT-proBNP. Myocardial hypertrophy and myocardial fibrosis were assessed by histological staining. ß-arr2, SERCA2a, SUMO1, PLB and p-PLB expressions were detected by Western blotting, immunofluorescence and immunohistochemistry. SERCA2a SUMOylation was detected by Co-IP. The molecular docking method was used to predict the binding ability of the main active components of QFYXF to ß-arr2, SERCA2a, and SUMO1, and the binding degree of SERCA2a to SUMO1 protein. Results: The HF model was constructed 8 weeks after TAC. QFYXF ameliorated cardiac function, inhibiting myocardial hypertrophy and fibrosis. QFYXF promoted SERCA2a expression and SERCA2a SUMOylation. Further investigation showed that QFYXF promoted ß-arr2 expression, whereas Barbadin (ß-arr2 inhibitor) or ß-arr2-KO reduced SERCA2a SUMOylation and attenuated the protective effect of QFYXF improved HF. Molecular docking showed that the main active components of QFYXF had good binding activities with ß-arr2, SERCA2a, and SUMO1, and SERCA2a had a high binding degree with SUMO1 protein. Conclusion: QFYXF improves HF by promoting ß-arr2 mediated SERCA2a SUMOylation and increasing SERCA2a expression.

Compound SJ-12 attenuates streptozocin-induced diabetic cardiomyopathy by stabilizing SERCA2a.

Heart failure (HF) is one of the major causes of death among diabetic patients. Although studies have shown that curcumin analog C66 can remarkably relieve diabetes-associated cardiovascular and kidney complications, the role of SJ-12, SJ-12, a novel curcumin analog, in diabetic cardiomyopathy and its molecular targets are unknown. 7-week-old male C57BL/6 mice were intraperitoneally injected with single streptozotocin (STZ) (160 mg/kg) to develop diabetic cardiomyopathy (DCM). The diabetic mice were then treated with SJ-12 via gavage for two months. Body weight, fast blood glucose, cardiac utrasonography, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers were assessed. The potential target of SJ-12 was evaluated via RNA-sequencing analysis. The O-GlcNAcylation levels of SP1 were detected via immunoprecipitation. SJ-12 effectively suppressed myocardial hypertrophy and fibrosis, thereby preventing heart dysfunction in mice with STZ-induced heart failure. RNA-sequencing analysis revealed that SJ-12 exerted its therapeutic effects through the modulation of the calcium signaling pathway. Furthermore, SJ-12 reduced the O-GlcNAcylation levels of SP1 by inhibiting O-linked N-acetylglucosamine transferase (OGT). Also, SJ-12 stabilized Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a (SERCA2a), a crucial regulator of calcium homeostasis, thus reducing hypertrophy and fibrosis in mouse hearts and cultured cardiomyocytes. However, the anti-fibrotic effects of SJ-12 were not detected in SERCA2a or OGT-silenced cardiomyocytes, indicating that SJ-12 can prevent DCM by targeting OGT-dependent O-GlcNAcylation of SP1.These findings indicate that SJ-12 can exert cardioprotective effects in STZ-induced mice by reducing the O-GlcNAcylation levels of SP1, thus stabilizing SERCA2a and reducing myocardial fibrosis and hypertrophy. Therefore, SJ-12 can be used for the treatment of diabetic cardiomyopathy.

Fructose aggravates copper-deficiency-induced cardiac remodeling by inhibiting SERCA2a.

OBJECTIVES: Accumulating evidence demonstrates that copper deficiency (CuD) is a risk factor for cardiovascular diseases, besides, fructose has been strongly linked to the development of cardiovascular diseases. However, how CuD or fructose causes cardiovascular diseases is not clearly delineated. The present study aims to investigate the mechanism of CuD or fructose on cardiac remodeling. METHODS: We established a model of CuD- or fructose-induced cardiac hypertrophy in 3-week-old male Sprague-Dawley (SD) rats by CuD diet supplemented with or without 30% fructose for 4 weeks. In vitro study was performed by treating cardiomyocytes with tetrathiomolydbate (TM) and fructose. Echocardiography, histology analysis, immunofluorescence, western blotting, and qPCR were performed. KEY FINDINGS: Our findings revealed that CuD caused noticeable cardiac hypertrophy either in the presence or absence of fructose supplement. Fructose exacerbated CuD-induced cardiac remodeling and intramyocardial lipid accumulation. Furthermore, we presented that the inhibition of autophagic flux caused by Ca2+ disturbance is the key mechanism by which CuD- or fructose-induced cardiac remodeling. The reduced expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) in cardiomyocytes accounts for the elevated cytoplasmic Ca2+ concentration. CONCLUSIONS: Collectively, our study suggested that fructose aggravated CuD-induced cardiac remodeling through the blockade of autophagic flux via SERCA2a decreasing-induced Ca2+ imbalance.

Selective SERCA2a activator as a candidate for chronic heart failure therapy.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized. METHODS: Effects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects). RESULTS: As expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity. CONCLUSIONS: The pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.

Early consequences of the phospholamban mutation PLN-R14del+/- in a transgenic mouse model.

AIMS: The heterozygous phospholamban (PLN) mutation R14del (PLN R14del+/- ) is associated with a severe arrhythmogenic cardiomyopathy (ACM) developing in the adult. "Superinhibition" of SERCA2a by PLN R14del is widely assumed to underlie the pathogenesis, but alternative mechanisms such abnormal energy metabolism have also been reported. This work aims to (1) to evaluate Ca2+ dynamics and energy metabolism in a transgenic (TG) mouse model of the mutation prior to cardiomyopathy development; (2) to test whether they are causally connected. METHODS: Ca2+ dynamics, energy metabolism parameters, reporters of mitochondrial integrity, energy, and redox homeostasis were measured in ventricular myocytes of 8-12 weeks-old, phenotypically silent, TG mice. Mutation effects were compared to pharmacological PLN antagonism and analyzed during modulation of sarcoplasmic reticulum (SR) and cytosolic Ca2+ compartments. Transcripts and proteins of relevant signaling pathways were evaluated. RESULTS: The mutation was characterized by hyperdynamic Ca2+ handling, compatible with a loss of SERCA2a inhibition by PLN. All components of energy metabolism were depressed; myocyte energy charge was preserved under quiescence but reduced during stimulation. Cytosolic Ca2+ buffering or SERCA2a blockade reduced O2 consumption with larger effect in the mutant. Signaling changes suggest cellular adaptation to perturbed Ca2+ dynamics and response to stress. CONCLUSIONS: (1) PLN R14del+/- loses its ability to inhibit SERCA2a, which argues against SERCA2a superinhibition as a pathogenetic mechanism; (2) depressed energy metabolism, its enhanced dependency on Ca2+ and activation of signaling responses point to an early involvement of metabolic stress in the pathogenesis of this ACM model.

SERCA2 dysfunction triggers hypertension by interrupting mitochondrial homeostasis and provoking oxidative stress.

BACKGROUND AND AIM: Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is critical in maintaining Ca2+ homeostasis. The cysteine 674 (C674) is the key redox regulatory cysteine in regulating SERCA2 activity, which is irreversibly oxidized in the renal cortex of hypertensive mice. We have reported that the substitution of C674 by serine causes SERCA2 dysfunction and increases blood pressure by induction of endoplasmic reticulum stress (ERS). This study is to explore whether the dysfunction of SERCA2 causes hypertension by interrupting mitochondrial homeostasis and inducing oxidative stress. METHODS & RESULTS: We used heterozygous SERCA2 C674S gene mutation knock-in (SKI) mice, where one copy of C674 was substituted by serine to represent partial C674 oxidation. In renal proximal tubule (RPT) cells, the substitution of C674 by serine decreased mitochondrial Ca2+ content, increased mitochondrial membrane potential, ATP content, and reactive oxygen species (ROS), which could be reversed by ERS inhibitor 4-phenylbutyric acid or SERCA2 agonist CDN1163. In SKI RPT cells, the redox modulator Tempol alleviated oxidative stress, downregulated the protein expression of ERS markers and soluble epoxide hydrolase, upregulated the protein expression of dopamine D1 receptor, and reduced Na+/K+- ATPase activity. In SKI mice, SERCA2 agonists CDN1163 and [6]-Gingerol, or the redox modulator Tempol increased urine output and lowered blood pressure. CONCLUSION: The irreversible oxidation of C674 is not only an indicator of increased ROS, but also further inducing oxidative stress to cause hypertension. Activation of SERCA2 or inhibition of oxidative stress is beneficial to alleviate hypertension caused by SERCA2 dysfunction.

Loss of STIM2 in colorectal cancer drives growth and metastasis through metabolic reprogramming and PERK-ATF4 endoplasmic reticulum stress pathway.

The endoplasmic reticulum (ER) stores large amounts of calcium (Ca2+), and the controlled release of ER Ca2+ regulates a myriad of cellular functions. Although altered ER Ca2+ homeostasis is known to induce ER stress, the mechanisms by which ER Ca2+ imbalance activate ER stress pathways are poorly understood. Stromal-interacting molecules STIM1 and STIM2 are two structurally homologous ER-resident Ca2+ sensors that synergistically regulate Ca2+ influx into the cytosol through Orai Ca2+ channels for subsequent signaling to transcription and ER Ca2+ refilling. Here, we demonstrate that reduced STIM2, but not STIM1, in colorectal cancer (CRC) is associated with poor patient prognosis. Loss of STIM2 causes SERCA2-dependent increase in ER Ca2+, increased protein translation and transcriptional and metabolic rewiring supporting increased tumor size, invasion, and metastasis. Mechanistically, STIM2 loss activates cMyc and the PERK/ATF4 branch of ER stress in an Orai-independent manner. Therefore, STIM2 and PERK/ATF4 could be exploited for prognosis or in targeted therapies to inhibit CRC tumor growth and metastasis.

CDN1163 alleviates SERCA2 dysfunction-induced pulmonary vascular remodeling by inhibiting the phenotypic transition of pulmonary artery smooth muscle cells.

BACKGROUND AND PURPOSE: Substitution of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes SERCA2 dysfunction which leads to activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway accelerating cell proliferation of pulmonary artery smooth muscle cells (PASMCs) followed by significant pulmonary vascular remodeling resembling human pulmonary hypertension. Based on this knowledge, we intend to investigate other potential mechanisms involved in SERCA2 dysfunction-induced pulmonary vascular remodeling. EXPERIMENTAL APPROACH: Heterozygous SERCA2 C674S knock-in (SKI) mice of which half of cysteine in 674 was substituted by serine to mimic the partial irreversible oxidation of C674 were used. The lungs of SKI mice and their littermate wild-type mice were collected for PASMC culture, protein expression, and pulmonary vascular remodeling analysis. RESULTS: SERCA2 dysfunction increased intracellular Ca2+ levels, which activated Ca2+-dependent calcineurin (CaN) and promoted the nuclear translocation and protein expression of the nuclear factor of activated T-lymphocytes 4 (NFAT4) in an IRE1α/XBP1s pathway-independent manner. In SKI PASMCs, the scavenge of intracellular Ca2+ by BAPTA-AM or inhibition of CaN by cyclosporin A can prevent PASMC phenotypic transition. CDN1163, a SERCA2 agonist, suppressed the activation of CaN/NFAT4 and IRE1α/XBP1s pathways, reversed the protein expression of PASMC phenotypic transition markers and cell cycle-related proteins, and inhibited cell proliferation and migration when given to SKI PASMCs. Furthermore, CDN1163 ameliorated pulmonary vascular remodeling in SKI mice. CONCLUSIONS AND IMPLICATIONS: SERCA2 dysfunction promotes PASMC phenotypic transition and pulmonary vascular remodeling by multiple mechanisms, which could be improved by SERCA2 agonist CDN1163.

'What is already known' l The dysfunction of SERCA2 promotes PASMC hyperproliferation and pulmonary vascular remodeling through activation of the IRE1α/XBP1s pathway.'What this study adds' l The dysfunction of SERCA2 activates the Ca²⁺-dependent CaN-mediated NFAT4 pathway to promote the PASMC phenotypic transition.l Revitalization of SERCA2 suppresses PASMC phenotypic transition and pulmonary vascular remodeling caused by SERCA2 dysfunction.'Clinical significance' l SERCA2 dysfunction-induced pulmonary vascular remodeling involves more than one mechanism, implicating that more drugable targets are to be discovered.l SERCA2 is a potential therapeutic target for preventing pulmonary vascular remodeling.

Polyarginine Cell-Penetrating Peptides Bind and Inhibit SERCA2.

Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.

Poly (ADP-ribose) polymerase pathway inhibitor (Olaparib) upregulates SERCA2a expression and attenuates doxorubicin-induced cardiomyopathy in mice.

The cardiotoxicity induced by doxorubicin is dose-dependent. The present study tested the potential cardioprotective effect of Poly ADP Ribose Polymerase (PARP) pathway inhibitor "olaparib" in a mouse model of doxorubicin-induced cardiomyopathy (DOX-CM). Seventy-two male BALB/c mice were randomized into six equal groups; control, DOX-CM, dexrazoxane-treated, and three olaparib-treated groups (5, 10, and 50 mg/kg/day). Cardiomyopathy was assessed by heart weight/Tibial length (HW/TL) ratio, cardiac fibrosis, oxidative stress, and electron microscope. Myocardial expression of SERCA2a mRNA and cleaved PARP-1 protein were also assessed. Similar to dexrazoxane, olaparib (10 mg/kg/day) significantly ameliorated oxidative stress, and preserved cardiac structure. It also suppressed myocardial PARP-1 protein expression and boosted SERCA2a mRNA expression. Olaparib (5 or 50 mg/kg/day) failed to show comparable effects. The current study detected the cardioprotective effect of olaparib at a dosage of 10 mg/kg/day. Also, the present study discovered a new cardioprotective mechanism of dexrazoxane by targeting PARP-1 in the heart.

Translocation of IGF-1R in endoplasmic reticulum enhances SERCA2 activity to trigger Ca2+ER perturbation in hepatocellular carcinoma.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rß is translocated into the ER by ß-arrestin2 (ß-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rß. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rß levels. ER IGF-1Rß phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rß with SERCA2, and therefore ER IGF-1Rß failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rß and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.

Therapeutic Aspects of Prunus cerasus Extract in a Rabbit Model of Atherosclerosis-Associated Diastolic Dysfunction.

This study evaluates the potential therapeutic effects of anthocyanin-rich Prunus cerasus (sour cherry) extract (PCE) on atherosclerosis-associated cardiac dysfunction, described by the impairment of the NO-PKG (nitric oxide-protein kinase G) pathway and the antioxidant capacity. Initially, a rabbit model of atherosclerotic cardiovascular disease was established by administering a cholesterol-rich diet, enabling the examination of the impact of 9 g/kg PCE on the pre-existing compromised cardiovascular condition. After that, the animals were divided into four groups for 12 weeks: the (1) untreated control group; (2) PCE-administered healthy rabbits; (3) hypercholesterolemic (HC) group kept on an atherogenic diet; and (4) PCE-treated HC group. Dyslipidemia, impaired endothelial function, and signs of diastolic dysfunction were evident in hypercholesterolemic rabbits, accompanied by a reduced cardiac expression of eNOS (endothelial nitric oxide synthase), PKG, and SERCA2a (sarco/endoplasmic reticulum calcium ATPase 2a). Subsequent PCE treatment improved the lipid profile and the cardiac function. Additionally, PCE administration was associated with elevated myocardial levels of eNOS, PKG, and SERCA2a, while no significant changes in the vascular status were observed. Western blot analysis further revealed hypercholesterolemia-induced increase and PCE-associated reduction in heme oxygenase-1 expression. The observed effects of anthocyanins indicate their potential as a valuable addition to the treatment regimen for atherosclerosis-associated cardiac dysfunction.

Termination codon readthrough of NNAT mRNA regulates calcium-mediated neuronal differentiation.

Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, cannot increase cytoplasmic Ca2+ levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca2+ levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.

SERCA2 phosphorylation at the heart of the disease.

Gonnot et al. [1] thoroughly investigated the regulatory role of glycogen synthase kinase 3 beta (GSK3ß) in modulating cardiac isoform 2 of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) activity. They have found that in ischemic hearts of patients and mouse-GSK3ß -mediated SERCA2 phosphorylation at serine 663 dampens the SERCA2 pump activity and induces Ca2+ overload which sensitizes towards myocardial ischemia-reperfusion (I/R) injury. The inhibition of serine 663 phosphorylation significantly increases SERCA2 activity and, by preventing cytosolic and mitochondrial Ca2+ overload, reduces cell death during reperfusion. Augmented SERCA2 activity also substantially improves excitation-contraction coupling in cardiomyocytes upon recovery from reperfusion injury. This study provides valuable insights into pathophysiological relevance of GSK3ß -mediated SERCA2 phosphorylation in the context of heart diseases and paves the way for designing novel clinical therapeutic approaches to alleviate post infartion heart failure.

Inhibition of miR-25 Ameliorates Cardiac Dysfunction and Fibrosis by Restoring Krüppel-like Factor 4 Expression.

Cardiac hypertrophy is an adaptive response to various pathological insults, including hypertension. However, sustained hypertrophy can cause impaired calcium regulation, cardiac dysfunction, and remodeling, accompanied by cardiac fibrosis. Our previous study identified miR-25 as a regulator of SERCA2a, and found that the inhibition of miR-25 improved cardiac function and reduced fibrosis by restoring SERCA2a expression in a murine heart failure model. However, the precise mechanism underlying the reduction in fibrosis following miR-25 inhibition remains unclear. Therefore, we postulate that miR-25 may have additional targets that contribute to regulating cardiac fibrosis. Using in silico analysis, Krüppel-like factor 4 (KLF4) was identified as an additional target of miR-25. Further experiments confirmed that KLF4 was directly targeted by miR-25 and that its expression was reduced by long-term treatment with Angiotensin II, a major hypertrophic inducer. Subsequently, treatment with an miR-25 inhibitor alleviated the cardiac dysfunction, fibrosis, and inflammation induced by Angiotensin II (Ang II). These findings indicate that inhibiting miR-25 not only enhances calcium cycling and cardiac function via SERCA2a restoration but also reduces fibrosis by restoring KLF4 expression. Therefore, targeting miR-25 may be a promising therapeutic strategy for treating hypertensive heart diseases.

Mettl13 protects against cardiac contractile dysfunction by negatively regulating C-Cbl-mediated ubiquitination of SERCA2a in ischemic heart failure.

Ischemic heart failure (HF) remains a leading cause of morbidity and mortality. Maintaining homeostasis of cardiac function and preventing cardiac remodeling deterioration are critical to halting HF progression. Methyltransferase-like protein 13 (Mettl13) has been shown to regulate protein translation efficiency by acting as a protein lysine methyltransferase, but its role in cardiac pathology remains unexplored. This study aims to characterize the roles and mechanisms of Mettl13 in cardiac contractile function and HF. We found that Mettl13 was downregulated in the failing hearts of mice post-myocardial infarction (MI) and in a cellular model of oxidative stress. Cardiomyocyte-specific overexpression of Mettl13 mediated by AAV9-Mettl13 attenuated cardiac contractile dysfunction and fibrosis in response to MI, while silencing of Mettl13 impaired cardiac function in normal mice. Moreover, Mettl13 overexpression abrogated the reduction in cell shortening, Ca2+ transient amplitude and SERCA2a protein levels in the cardiomyocytes of adult mice with MI. Conversely, knockdown of Mettl13 impaired the contractility of cardiomyocytes, and decreased Ca2+ transient amplitude and SERCA2a protein expression in vivo and in vitro. Mechanistically, Mettl13 impaired the stability of c-Cbl by inducing lysine methylation of c-Cbl, which in turn inhibited ubiquitination-dependent degradation of SERCA2a. Furthermore, the inhibitory effects of knocking down Mettl13 on SERCA2a protein expression and Ca2+ transients were partially rescued by silencing c-Cbl in H2O2-treated cardiomyocytes. In conclusion, our study uncovers a novel mechanism that involves the Mettl13/c-Cbl/SERCA2a axis in regulating cardiac contractile function and remodeling, and identifies Mettl13 as a novel therapeutic target for ischemic HF.

Lysine crotonylation of SERCA2a correlates to cardiac dysfunction and arrhythmia in Sirt1 cardiac-specific knockout mice.

Protein post-translational modifications (PTMs) are important regulators of protein functions and produce proteome complexity. SIRT1 has NAD+-dependent deacylation of acyl-lysine residues. The present study aimed to explore the correlation between lysine crotonylation (Kcr) on cardiac function and rhythm in Sirt1 cardiac-specific knockout (ScKO) mice and related mechanism. Quantitative proteomics and bioinformatics analysis of Kcr were performed in the heart tissue of ScKO mice established with a tamoxifen-inducible Cre-loxP system. The expression and enzyme activity of crotonylated protein were assessed by western blot, co-immunoprecipitation, and cell biology experiment. Echocardiography and electrophysiology were performed to investigate the influence of decrotonylation on cardiac function and rhythm in ScKO mice. The Kcr of SERCA2a was significantly increased on Lys120 (1.973 folds). The activity of SERCA2a decreased due to lower binding energy of crotonylated SERCA2a and ATP. Changes in expression of PPAR-related proteins suggest abnormal energy metabolism in the heart. ScKO mice had cardiac hypertrophy, impaired cardiac function, and abnormal ultrastructure and electrophysiological activities. We conclude that knockout of SIRT1 alters the ultrastructure of cardiac myocytes, induces cardiac hypertrophy and dysfunction, causes arrhythmia, and changes energy metabolism by regulating Kcr of SERCA2a. These findings provide new insight into the role of PTMs in heart diseases.