Inhibition Of Cardiomyocytes Research Articles

Co-localization of the sodium-glucose co-transporter-2 channel (SGLT-2) with endothelin ETA and ETB receptors in human cardiorenal tissue.

Immunocytochemistry localised SGLT-2, ETA and ETB receptors in sections of histologically normal kidney, left ventricle from patients undergoing heart transplantation or controls. Primary antisera were visualised using fluorescent microscopy. Image analysis was used to measure intensity compared with background in adjacent control sections. As expected, SGLT-2 localised to epithelial cells of the proximal convoluted tubules, and co-localised with both ET receptor sub-types. Similarly, ETA receptors predominated in cardiomyocytes; low (compared with kidney but above background) positive staining was also detected for SGLT-2. Whether low levels of SGLT-2 have a (patho)physiological role in cardiomyocytes is not known but results suggest the effect of direct blockade of sodium (and glucose) influx via SGLT-2 inhibition in cardiomyocytes should be explored, with potential for additive effects with ETA antagonists.

Exosomes derived from cardiac fibroblasts with angiotensin II stimulation provoke hypertrophy and autophagy inhibition in cardiomyocytes

Although accumulating evidence has revealed that autophagy inhibition contributes to the development of pathological cardiac hypertrophy, the mechanisms leading to declined autophagy activity in the hypertrophic heart remain to be elucidated. Exosomes are known to be important mediators of intercellular communication, and the involvement of exosomes in cardiovascular abnormities has attracted increasing attentions. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. Here, we investigated the potential role of CFs-derived exosomes in regulating cardiomyocyte hypertrophy and autophagy. Exosomes from rat CFs treated with angiotensin II (Ang II-CFs-exosomes) were collected and characterized. Our experiments showed that these exosomes could induce hypertrophic responses and impair autophagy activity in primary neonatal rat cardiomyocytes (NRCMs). Ang II-CFs-exosomes blocked the autophagic flux of NRCMs via inhibiting the formation of autolysosomes. Moreover, the pro-hypertrophic effects and autophagy inhibition induced by Ang II-CFs-exosomes was validated in mice receiving injection of the exosomes. These findings highlight a novel role of Ang II-CFs-exosomes in suppressing cardiomyocyte autophagy, which may help to better understand the pathogenesis of cardiac hypertrophy.

Circular RNA FEACR inhibits ferroptosis and alleviates myocardial ischemia/reperfusion injury by interacting with NAMPT

BackgroundEmerging research has reported that circular RNAs (circRNAs) play important roles in cardiac cell death after myocardial ischemia and reperfusion (I/R). Ferroptosis, a new form of cell death discovered in recent years, has been proven to participate in the regulation of myocardial I/R. This study used circRNA sequencing to explore the key circRNA in the regulation of cardiac ferroptosis after I/R and study the mechanisms of potential circRNA function.MethodsWe performed circRNA sequencing to explore circRNAs differentially expressed after myocardial I/R. We used quantitative polymerase chain reactions to determine the circRNA expression in different tissues and detect the circRNA subcellular localization in the cardiomyocyte. Gain- and loss-of-function experiments were aimed to examine the function of circRNAs in cardiomyocyte ferroptosis and cardiac tissue damage after myocardial I/R. RNA pull-down was applied to explore proteins interacting with circRNA.ResultsHere, we identified a ferroptosis-associated circRNA (FEACR) that has an underlying regulatory role in cardiomyocyte ferroptosis. FEACR overexpression suppressed I/R-induced myocardial infarction and ameliorated cardiac function. FEACR inhibition induces ferroptosis in cardiomyocytes and FEACR overexpression inhibits hypoxia and reoxygenation-induced ferroptosis. Mechanistically, FEACR directly bound to nicotinamide phosphoribosyltransferase (NAMPT) and enhanced the protein stability of NAMPT, which increased NAMPT-dependent Sirtuin1 (Sirt1) expression, which promoted the transcriptional activity of forkhead box protein O1 (FOXO1) by reducing FOXO1 acetylation levels. FOXO1 further upregulated the transcription of ferritin heavy chain 1 (Fth1), a ferroptosis suppressor, which resulted in the inhibition of cardiomyocyte ferroptosis.ConclusionsOur finding reveals that the circRNA FEACR-mediated NAMPT-Sirt1-FOXO1-FTH1 signaling axis participates in the regulation of cardiomyocyte ferroptosis and protects the heart function against I/R injury. Thus, FEACR and its downstream factors could be novel targets for alleviating ferroptosis-related myocardial injury in ischemic heart diseases.

Loss of circIGF1R Suppresses Cardiomyocytes Proliferation by Sponging miR-362-5p.

Circular RNAs (circRNAs) are generally formed by the back-splicing of precursor mRNA. Increasing evidence implicates the important role of circRNAs in cardiovascular diseases. However, the role of circ-insulin-like growth factor 1 receptor (circIGF1R) in cardiomyocyte (CM) proliferation remains unclear. Here, we investigated the potential role of the circIGF1R in the proliferation of CMs. We found that circIGF1R expression in heart tissues and primary CMs from adult mice was significantly lower than that in neonatal mice at postnatal 1 day (p1). Increased circIGF1R expression was detected in the injured neonatal heart at 0.5 and 1 days post-resection. circIGF1R knockdown significantly decreased the proliferation of primary CMs. Combined prediction software, luciferase reporter gene analysis, and quantitative real time-PCR (qPCR) revealed that circIGF1R interacted with miR-362-5p. A significant increase in miR-362-5p expression was detected in the adult heart compared with that in the neonatal heart. Further, heart injury significantly decreased the expression of miR-362-5p in neonatal mice. Treatment with miR-362-5p mimics significantly suppressed the proliferation of primary CMs, whereas knockdown of miR-362-5p promoted the CMs proliferation. Meanwhile, miR-362-5p silencing can rescue the proliferation inhibition of CMs induced by circIGF1R knockdown. Target prediction and qPCR validation revealed that miR-362-5p significantly inhibited the expression of Phf3 in primary CMs. In addition, decreased Phf3 expression was detected in adult hearts compared with neonatal hearts. Consistently, increased Phf3 expression was detected in injured neonatal hearts compared with that in sham hearts. Knockdown of Phf3 markedly repressed CMs proliferation. Taken together, these findings suggest that circIGF1R might contribute to cardiomyocyte proliferation by promoting Pfh3 expression by sponging miR-362-5p and provide an important experimental basis for the regulation of heart regeneration.

Levosimendan Reverses Cardiac Malfunction and Cardiomyocyte Ferroptosis During Heart Failure with Preserved Ejection Fraction via Connexin 43 Signaling Activation.

In recent decades, the occurrence of heart failure with preserved ejection fraction (HFpEF) has outweighed that of heart failure with reduced ejection fraction by degrees, but few drugs have been demonstrated to improve long-term clinical outcomes in patients with HFpEF. Levosimendan, a calcium-sensitizing cardiotonic agent, improves decompensated heart failure clinically. However, the anti-HFpEF activities of levosimendan and underlying molecular mechanisms are unclear. In this study, a double-hit HFpEF C57BL/6N mouse model was established, and levosimendan (3 mg/kg/week) was administered to HFpEF mice aged 13 to 17 weeks. Different biological experimental techniques were used to verify the protective effects of levosimendan against HFpEF. After four weeks of drug treatment, left ventricular diastolic dysfunction, cardiac hypertrophy, pulmonary congestion, and exercise exhaustion were significantly alleviated. Junction proteins in the endothelial barrier and between cardiomyocytes were also improved by levosimendan. Among the gap junction channel proteins, connexin 43, which was especially highly expressed in cardiomyocytes, mediated mitochondrial protection. Furthermore, levosimendan reversed mitochondrial malfunction in HFpEF mice, as evidenced by increased mitofilin and decreased ROS, superoxide anion, NOX4, and cytochrome C levels. Interestingly, after levosimendan administration, myocardial tissue from HFpEF mice showed restricted ferroptosis, indicated by an increased GSH/GSSG ratio; upregulated GPX4, xCT, and FSP-1 expression; and reduced intracellular ferrous ion, MDA, and 4-HNE levels. Regular long-term levosimendan administration can benefit cardiac function in a mouse model of HFpEF with metabolic syndromes (namely, obesity and hypertension) by activating connexin 43-mediated mitochondrial protection and sequential ferroptosis inhibition in cardiomyocytes.

Inhibition of HIPK2 protects stress-induced pathological cardiac remodeling.

SummaryBackgroundHomeodomain-Interacting Protein Kinase 2 (HIPK2) has been reported to maintain basal cardiac function, however, its role in pathological cardiac remodeling remains unclear.MethodsHIPK2 inhibitors (tBID and PKI1H) treated mice and two lines of HIPK2−/− mice were subjected to transverse aortic constriction (TAC). HIPK2 knockdown were performed in neonatal rat cardiomyocytes (NRCMs), neonatal rat cardiac fibroblasts (NRCFs), and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Microarray analysis was used to screen HIPK2 targets. Overexpression of early growth response 3 (EGR3) and C-type lectin receptor 4D (CLEC4D) were performed in NRCMs, while an activator of Smad3 was used in NRCFs, to rescue the effects of HIPK2 knockdown. Finally, the effects of EGR3 and CLEC4D knockdown by AAV9 in TAC were determined.FindingsHIPK2 was elevated in TAC mice model, as well as cardiomyocyte hypertrophy and NRCFs fibrosis model. Pharmacological and genetic inhibition of HIPK2 improved cardiac function and suppressed cardiac hypertrophy and fibrosis induced by TAC. In vitro, HIPK2 inhibition prevented cardiomyocyte hypertrophic growth and NRCFs proliferation and differentiation. At the mechanistic level, we identified EGR3 and CLEC4D as new targets of HIPK2, which were regulated by ERK1/2-CREB and mediated the protective function of HIPK2 inhibition in cardiomyocytes. Meanwhile, inhibition of phosphorylation of Smad3 was responsible for the suppression of cardiac fibroblasts proliferation and differentiation by HIPK2 inhibition. Finally, we found that inhibition of EGR3 or CLEC4D protected against TAC.InterpretationHIPK2 inhibition protects against pathological cardiac remodeling by reducing EGR3 and CLEC4D with ERK1/2-CREB inhibition in cardiomyocytes, and by suppressing the phosphorylation of Smad3 in cardiac fibroblasts.FundingThis work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J.X.), National Natural Science Foundation of China (82020108002 and 81911540486 to J.X., 81400647 to MJ Xu), the grant from Science and Technology Commission of Shanghai Municipality (21XD1421300 and 20DZ2255400 to J.X.), the “Dawn” Program of Shanghai Education Commission (19SG34 to J.X.), and Shanghai Sailing Program (21YF1413200 to Q.Z.).

EGFR Inhibition in Cardiomyocytes Stabilizes Cardiomyocyte Cohesion in a Murine Model for Arrhythmogenic Cardiomyopathy

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase ubiquitously expressed in different tissues, including the heart. EGFR is dysregulated in several kinds of cancer, as well as in heart diseases. Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease and one of the underlying mechanisms is impaired desmosome turnover. The intercalated disc (ICD) not only provides cellular cohesion but can also provide the structural framework of a signaling hub. Thus, stabilizing desmosome integrity, thereby enhancing cardiomyocyte stability, may provide potential new treatment options. In this study, we investigated the role of EGFR inhibition on cardiomyocyte cohesion under physiological as well as under pathophysiological conditions using the cardiomyocyte‐specific plakoglobin knockout (Jup‐/‐) ACM model in which EGFR was upregulated. Dissociation assays in HL‐1 cells and murine cardiac slice cultures showed that EGFR inhibition by erlotinib similar to SRC inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR, desmoglein 2 (DSG2) and desmoplakin (DP), indicating that altered EGFR signaling might affect desmosomes. Immunostainings and atomic force microscopy (AFM) revealed enhanced DSG2 localization and binding at the cell borders upon EGFR inhibition. In addition, during desmosome assembly induced by a Ca2+‐switch, DSG2 and DP were augmented at the cell borders after EGFR or SRC inhibition. Our study shows that EGFR inhibition by erlotinib leads to increased DSG2 binding and desmosome assembly, which stabilizes cardiomyocyte cohesion in the ACM mouse model. Because EGFR inhibition by erlotinib is already used in cancer patients, these data reveal a potential new therapeutic strategy in ACM.

Cardiovascular toxic effects of antitumor agents: Pathogenetic mechanisms

Cancer treatment is associated with various side effects of antitumor agents, which increase the morbidity and mortality of these patients. Cardiovascular complications are considered to be one of the most important side effect of the anticancer drugs. The antitumor drugs that express cardiovascular effects include anthracyclines, tyrosine kinase inhibitors, taxanes, fluoropyrimidines, alkylating agents, vascular endothelial growth factor inhibitors, immune checkpoint inhibitors, proteasome inhibitors and human epidermal growth receptor type 2 antibodies. The spectrum of cardiovascular effects of anticancer drugs is broad and include, among others, heart failure, arrhythmias such as atrial fibrillation and ventricular tachyarrhythmias, hypertension (systemic or pulmonary), cardiomyopathy, myocarditis, valve disease, pericardial disease, vascular events (arterial thrombosis, venous thromboembolism) and myocardial ischemia (acute coronary syndrome, angina). The molecular mechanisms by which anti-cancer therapies lead to cardiotoxicity are diverse and vary according to the specific type of agent used. They include oxidative stress, topoisomerase 2-β inhibition in cardiomyocytes, inflammation, endothelial dysfunction, apoptosis, disruption of Ca2+ homeostasis, mitochondrial dysfunction, DNA damage, increase in various circulating microRNAs levels, alterations in the function of voltage-gated potassium channels. The management of cardiovascular complications in cancer patients is a new challenge for oncologists and cardiologists. Thus the cardio-oncology field has developed the last decade in order to precisely predict and efficiently treat the cancer treatment-related cardiovascular diseases.

MicroRNA-146b-5p promotes atrial fibrosis in atrial fibrillation by repressing TIMP4.

Alteration of tissue inhibitors of matrix metalloproteinases (TIMP)/matrix metalloproteinases (MMP) associated with collagen upregulation has an important role in sustained atrial fibrillation (AF). The expression of miR‐146b‐5p, whose the targeted gene is TIMPs, is upregulated in atrial cardiomyocytes during AF. This study was to determine whether miR‐146b‐5p could regulate the gene expression of TIMP4 and the contribution of miRNA to atrial fibrosis in AF. Collagen synthesis was observed after miR‐146b‐5p transfection in human induced pluripotent stem cell‐derived atrial cardiomyocytes (hiPSC‐aCMs)‐fibroblast co‐culture cellular model in vitro. Furthermore, a myocardial infarction (MI) mouse model was used to confirm the protective effect of miR‐146b‐5p downregulation on atrial fibrosis. The expression level of miR‐146b‐5p was upregulated, while the expression level of TIMP4 was downregulated in the fibrotic atrium of canine with AF. miR‐146b‐5p transfection in hiPSC‐aCMs‐fibroblast co‐culture cellular model increased collagen synthesis by regulating TIMP4/MMP9 mediated extracellular matrix proteins synthesis. The inhibition of miR‐146b‐5p expression reduced the phenotypes of cardiac fibrosis in the MI mouse model. Fibrotic marker MMP9, TGFB1 and COL1A1 were significantly downregulated, while TIMP4 was significantly upregulated (at both mRNA and protein levels) by miR‐146b‐5p inhibition in cardiomyocytes of MI heart. We concluded that collagen fibres were accumulated in extracellular space on miR‐146b‐5p overexpressed co‐culture cellular model. Moreover, the cardiac fibrosis induced by MI was attenuated in antagomiR‐146 treated mice by increasing the expression of TIMP4, which indicated that the inhibition of miR‐146b‐5p might become an effective therapeutic approach for preventing atrial fibrosis.

P-158: Carfilzomib-induced acute cardiotoxicity is mediated through angiotensin and caused by cardiomyocyte energy depletion

<h3>Background</h3> Carfilzomib (CFZ) treatment increases the survival of patients with relapsed/refractory MM, but it is associated with a higher incidence of cardiovascular adverse events not commonly observed after bortezomib (BTZ). Both CFZ and BTZ inhibit the rate-limiting proteasome beta 5 subunit activity at lower concentrations. However, only CFZ co-inhibits the activity of proteasome beta 5 and beta 2 subunits at higher doses, in contrast to the beta 5 and beta 1 co-inhibition provided by high dose BTZ. We hypothesized that the unique beta 5 and beta 2 subunit inhibition pattern explains the CFZ-related acute cardiotoxicity. <h3>Methods</h3> Isolated primary murine cardiomyocytes treated with BTZ, CFZ or specific inhibitors for the beta 5, beta 2 and beta 1 proteasome subunits were used as in vitro model to assess cardiomyocyte contractility by time lapse video-microscopy in conjunction with motion vector image analysis. The effects of acute proteasome inhibition after CFZ or BTZ treatment was studied in vivo in the heart and bone marrow 1 hour post treatment with a multi-omics approach at the single cell-genomic, proteomic and metabolomic level. Selectivity of proteasome subunit inhibition was confirmed with the activity based chemical probes. All data was evaluated with R v.3.5.1 (2018-07-02) and Matlab 2020a. <h3>Results</h3> CFZ resulted in co-inhibition of beta 5 and beta 2 proteasome subunit activity in cardiomyocytes, in contrast to beta 5 and beta 1 inhibition by BTZ. CFZ, or the combination of beta 5 and beta 2 specific proteasome inhibitors induced acute impairment of cardiomyocyte contractility, in contrast to BTZ or the combination of beta 5 and beta 1 inhibitors. CFZ caused significant quantitative proteomic changes related to cardiomyocyte metabolism that differed from BTZ-related proteomic changes. Single-cell RNA sequencing of murine hearts revealed that CFZ impairs ATP synthesis in cardiomyocytes. Metabolomic analysis of murine hearts revealed increased levels of angiotensin after CFZ treatment, in contrast to BTZ treatment. Co-treatment of cardiomyocytes with CFZ and valsartan prevented CFZ-induced cardiomyocyte toxicity. Thus, our results suggest a key role of the renin-angiotensin system in the pathogenesis of CFZ-induced acute cardiac toxicity. <h3>Conclusion</h3> Our data suggest that CFZ specifically impairs cardiac contractility in a dose-dependent manner through its unique proteasome beta 5 and beta 2 subunit co-inhibition pattern, in contrast to BTZ. CFZ leads to more efficient functional proteasome inhibition in cardiomyocytes, impairs cardiomyocyte metabolism and activates the local renin-angiotensin-system, inducing acute cardiotoxicity. Inhibition of the renin-angiotensin system pathway with valsartan mitigated cardiomyocyte contractile impairment, providing a rational strategy to prevent CFZ-induced acute cardiac toxicity.

The Role of GSK3β in SERCA Dysfunction and the Development of Arrhythmogenic Cardiomyopathy

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump catalyzes the active transport of Ca2+ into the sarcoplasmic reticulum and is responsible for over 70% of Ca2+ turnover after muscle contraction. Phospholamban (PLN) is a well-known SERCA regulator, and impaired SERCA function due to reductions in SERCA and/or enhanced inhibitory PLN action has been linked to the development of various cardiomyopathies. Arrhythmogenic cardiomyopathy (ACM) is an inherited, non-ischemic heart disease presenting with ventricular arrhythmias, hypo/dyskinesia, and fibrotic remodeling. Over 60% of ACM cases are caused by pathogenic variants in genes encoding the cardiac desmosome, where Desmoglein-2 (DSG2) is the second most prevalent desmosomal gene variant in the ACM patient population. Using Dsg2 mutant (Dsg2mut/mut) mice, previous studies showed that the enzyme glycogen synthase kinase-3β (GSK3β), plays a major role in ACM pathogenesis. Inhibiting GSK3β in these mice improved cardiac contractility and ACM pathological phenotypes. We have shown that GSK3β inhibition in cardiomyocytes can also improve SERCA function in wild-type (WT) mice; however, the role of GSK3β activity on SERCA function in Dsg2mut/mut mice is currently unknown. Here, we sought to examine the effects of GSK3β inhibition on SERCA function using a GSK3β-specific inhibitor, SB216763 (SB2), in Dsg2mut/mut mice. At 3 weeks of age, both WT and Dsg2mut/mut mice were treated with SB2 (2.5 mg/kg/day) or equivalent volume/kg/day of vehicle (DMSO) for 13 weeks via intraperitoneal injection. After treatment, mice were sacrificed and hearts were collected and homogenized. Our results show that cardiomyocytes obtained from vehicle treated Dsg2mut/mut mice had significantly lower SERCA2a expression (-52%, p<0.01) compared to vehicle treated WT mice, leading to reduced SERCA2a:PLN (-65%, p=0.04). Maximal SERCA activity was also significantly lower in vehicle treated Dsg2mut/mut mice (-65%, p=0.001). However, in myocardium from SB2 treated Dsg2mut/mut mice, maximal SERCA activity, SERCA2a expression, and SERCA2a:PLN ratio were all restored to near WT levels. We also examined the effects of heterozygous and homozygous GSK3β S9A mutation in the Dsg2mut/mut mice, which prevents phosphorylation and maintains GSK3β in its constitutively active form. When comparing WT with Dsg2mut/mutheterozygous and homozygous GSK3β S9A mutants, we saw a significant but non-progressive decrease in maximal SERCA activity (-60%, p=0.02 and -58%, p=0.03 respectively). In conclusion, these results show that SERCA function is negatively impacted in Dsg2mut/mut cardiomyocytes. Conversely, GSK3β inhibition with SB2 treatment improved SERCA content and activity to healthy WT levels. These results further illuminate the role of GSK3β in ACM pathogenesis and support the use of GSK3β inhibitors in its treatment, as our new promising results indicate this therapeutic strategy can also improve Ca2+ handling.

Donepezil induces ventricular arrhythmias by delayed repolarisation.

Acetylcholinesterase inhibitors such as donepezil delay the progression of Alzheimer's dementia by increasing acetylcholine concentrations in the central nervous system. However, it is becoming apparent that cholinesterase inhibition by donepezil is not confined to the brain. This is supported by previous case reports of peripheral cholinergic side effects and adverse cardiac arrhythmias such as Torsades de Pointes which are reversible upon cessation of donepezil. The augmented acetylcholine concentrations and IKr inhibition in cardiomyocytes caused by donepezil are believed to mediate this effect.

Upregulation of miRNA-23a-3p rescues high glucose-induced cell apoptosis and proliferation inhibition in cardiomyocytes

Maternal hyperglycemia potentially inhibits the development of the fetal heart by suppressing cardiomyocyte proliferation and promoting apoptosis. Different studies have indicated that miRNAs are key regulators of cardiomyocyte proliferation, differentiation, and apoptosis and play a protective role in a variety of cardiovascular diseases. However, the biological function of miRNA-23a in hyperglycemia-related cardiomyocyte injury is not fully understood. The present study investigated the effect of miRNA-23a-3p on cell proliferation and apoptosis in a myocardial injury model induced by high glucose. H9c2 cardiomyocytes were exposed to high glucose to establish an in vitro myocardial injury model and then transfected with miRNA-23a-3p mimics. After miRNA-23a-3p transfection, lens-free microscopy was used to dynamically monitor cell numbers and confluence and calculate the cell cycle duration. CCK-8 and EdU incorporation assays were performed to detect cell proliferation. Flow cytometry was used to measured cell apoptosis. Upregulation of miRNA-23a-3p significantly alleviated high glucose-induced cell apoptosis and cell proliferation inhibition (p < 0.01 and p < 0.0001, respectively). The cell cycle of the miRNA-23a-3p mimics group was significantly shorter than that of the negative control group (p < 0.01). The expression of cell cycle–activating and apoptosis inhibition-associated factors Ccna2, Ccne1, and Bcl-2 was downregulated by high glucose and upregulated by miRNA-23a-3p overexpression in high glucose-injured H9c2 cells. miRNA-23a-3p mimics transfection before high glucose treatment had a significantly greater benefit than transfection after high glucose treatment (p < 0.0001), and the rescue effect of miRNA-23a-3p increased as the concentration increased. This study suggests that miRNA-23a-3p exerted a dose- and time-dependent protective effect on high glucose-induced H9c2 cardiomyocyte injury.

All-Trans-Retinoic Acid Prevents Carfilzomib-Induced Cardiotoxicity By Decreasing Activation of the Renin-Angiotensin System

INTRODUCTION Bortezomib (BTZ), Carfilzomib (CFZ) and Ixazomib (IXA) are proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment. By design, they all target the rate-limiting proteasome ß5 subunit. CFZ treatment increases the survival of patients with relapsed/refractory MM compared to BTZ. However, CFZ treatment is associated with heart failure not commonly observed for BTZ. The molecular basis for CFZ-induced cardiotoxicity is poorly understood. We have shown before that CFZ co-inhibits the ß5 and ß2 proteasome subunits compared to co-inhibition of the ß5/ß1 subunits by BTZ. We hypothesized that the unique ß5/ß2 subunit inhibition pattern of CFZ likewise explains the CFZ related acute cardiotoxicity. METHODS Isolated primary murine cardiomyocytes treated with BTZ, CFZ or ß5, ß2 and ß1 subunit-selective PI were used as in vitro model. Selectivity of proteasome subunit inhibition was confirmed with activity based chemical probes. Cardiomyocyte contractility was assessed in vitro using motion vector image analysis. The accumulation of individual proteins in cardiomyocytes undergoing 1h treatment with different PI was analyzed by liquid chromatography/mass spectrometry (LC/MS). In vivo analysis of the effects of differential proteasome subunit inhibition patterns was performed by electrocardiography (ECG) monitoring and untargeted metabolomics analysis from murine heart and plasma using R v.3.5.1 (2018-07-02) and Matlab 2020a. RESULTS Co-inhibition of ß5/ß2 proteasome subunits by CFZ or by respective subunit-specific PI for 1h resulted in increased proteasome inhibition and proteotoxic stress in cardiomyocytes compared to ß5/ß1 inhibition induced by BTZ. Likewise, CFZ or the ß5/ß2 type co-inhibition of the proteasome impaired contractility of cardiomyocytes in vitro, in contrast to BTZ or to ß5/ß1-selective proteasome inhibition. LC/MS-based proteome analysis of murine primary cardiomyocytes treated with either CFZ-type or BTZ-type proteasome inhibition identified accumulation of Retinal Dehydrogenase 1 (ALDH1A1) in PI-treated cardiomyocytes, with CFZ showing a slightly stronger effect. ALDH1A1 is involved in retinoic acid metabolism. Co-treatment of cardiomyocytes with its enzymatic end-product, all-trans-Retinoic-Acid (atRA), restored contractility of cardiomyocytes undergoing CFZ treatment in vitro. Leucyl-Cistinyl Aminopeptidase (LCAP) was identified as the protein that accumulated in CFZ-versus BTZ-treated cardiomyocytes (Log2 CFZ = 1.1, Log2 BTZ = -0.49, p=0.002). LCAP is involved in the Renin-Angiotensin System (RAS) and its increase suggests selective activation of RAS with increased degradation of RAS metabolites via LCAP in cardiomyocytes undergoing CFZ treatment. Indeed, mRNA expression for angiotensin converting enzyme 2 (Ace2) and angiotensin receptor 1 (Agtr1) was increased in beating cardiomyocytes treated with CFZ. Conversely, treatment with aminopeptidase inhibitor Bestatin prevented CFZ-induced cardiomyocyte toxicity in vitro. Consistent with our model, co-treatment of cardiomyocytes with CFZ and Valsartan, an Agtr1 inhibitor, likewise prevented CFZ- induced toxicity. In vivo, 1h CFZ treatment selectively led to acute bradycardia with local activation of the RAS and accumulation of angiotensin in the heart. CFZ/atRA co-treatment decreased local angiotensin levels and prevented CFZ-induced bradycardia. There were no differences in angiotensin plasma levels in between the treated mice, suggesting that the local RAS in the heart rather than systemic effects of the RAS mediates the cardiotoxicity of CFZ. CONCLUSION Our data suggest that CFZ specifically impairs cardiac contractility in a dose-dependent manner that is caused by its unique ß5/ß2 proteasome subunit co-inhibition pattern. CFZ leads to more efficient functional proteasome inhibition in cardiomyocytes, resulting in accumulation of LCAP that subsequently activates the local RAS and induce acute cardiotoxicity. Based on our findings, CFZ-induced cardiotoxicity may be prevented or mitigated by either blocking RAS activation at the angiotensin II receptor level using selective drugs like Valsartan, or by reducing angiotensin levels with atRA. This provides a rational strategy to prevent CFZ-induced acute cardiac toxicity by modulating the activity of the local RAS with approved drugs. Disclosures No relevant conflicts of interest to declare.

PRKAR1A deficiency delays postnatal heart growth

AimsProtein kinase A (PKA) activity is pivotal for proper functioning of the human heart, and its dysregulation has been implicated in a variety of cardiac pathologies. PKA regulatory subunit 1α (R1α, encoded by the PRKAR1A gene) is highly expressed in the heart, and controls PKA kinase activity by sequestering the catalytic subunits. Patients with PRKAR1A mutations are often diagnosed with Carney complex (CNC) in young adults and may die prematurely from cardiac complications such as heart failure. However, no cardiac defect has been reported in adult animal models of PRKAR1A deficiency.Methods and ResultsTo investigate the impact of PRKAR1A deficiency on the heart, we generated cardiac‐specific PRKAR1A heterozygous knockout (cPRKAR1A+/−) mice by breeding the floxed PRKAR1A mice with the Mlc2v‐Cre mice. We also studied a cohort of young CNC patients with PRKAR1A mutations or deletions. Ablation of the PRKAR1A gene in mice increased cardiac PKA activity, reduced heart weight to body weight ratio (Control: 5.3±0.19, cPRKAR1A+/−: 4.7±0.11 mg/g, P&lt;0.05) and cardiomyocyte size (Control: 298.5±9.8 μm2, cPRKAR1A+/−: 191.3±4.5 μm2, P&lt;0.005) without altering contractile function at 3 months of age. Importantly, left ventricular mass was reduced in young patients diagnosed with CNC (Female control: 80.6 ± 12.8g, CNC: 70.8 ± 8.1, P&lt;0.05). Cardiomyocyte hypertrophy in response to activation of the α1‐adrenergic receptor, which is necessary for heart growth after birth, was completely abolished by silencing of PRKAR1A, or stimulation with the PKA activator forskolin. Mechanistically, depletion of PRKAR1A provoked PKA‐dependent phosphorylation of the mitochondrial fission protein Drp1 at S637, leading to suppression of mitochondrial fission and inhibition of cardiomyocyte hypertrophy.ConclusionsCombined with previous findings that mitochondrial fission is required for hypertrophic growth of cardiomyocytes, our results suggest that PRKAR1A deficiency impedes postnatal myocardial development and physiological hypertrophy through modulation of mitochondrial dynamics. These findings provide a potential novel mechanism for the cardiac manifestations associated with CNC.Support or Funding InformationThis study was supported by intramural program of National Institute of Child Health and Human Development (NICHD), National Institutes of Health, National Heart, Lung, and Blood Institute (NIH R00HL119605, R56HL145034); WSU College of Pharmacy and Pharmaceutical Sciences.

In This Issue

HomeCirculation ResearchVol. 125, No. 1In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published20 Jun 2019https://doi.org/10.1161/RES.0000000000000281Circulation Research. 2019;125:2is related toRelationship Between Serum Alpha-Tocopherol and Overall and Cause-Specific Mortalityis related toMitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial CellsMacrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating InflammationSerum Alpha-Tocopherol and Mortality (p 29)A 30-year study suggests vitamin E has long-term health benefits, say Huang et al.Vitamin E, or α-tocopherol, is an essential fat-soluble vitamin acquired via consumption of vegetable oils, nuts, seeds, whole grains, and certain fruits and vegetables. With reported antioxidant, anti-inflammatory, and tumor-suppressive activities, α-tocopherol has apparent health benefits. But, while a lower risk of all-cause mortality has been linked to higher α-tocopherol levels in people, studies show inconsistent links with cause-specific deaths, such as those from cancer and cardiovascular disease. To look more closely at cause-specific mortality, Huang and colleagues have studied a cohort of almost 30,000 Finnish men who were in their 50s and 60s at the start of a 30-year health study and who had α-tocopherol levels, among other metrics, measured at baseline. From ≈24,000 deaths, and after adjusting for age and major risk factors such as smoking, the team found that α-tocopherol levels were inversely associated with the risk of death from cardiovascular disease, heart disease, stroke, cancer, respiratory disease, other-causes and all-causes, but not with risk of death from diabetes or injuries and accidents. The authors say these results indicate vitamin E may influence longevity, but also highlight the need for further studies in ethnically diverse populations and women.Stressed Mitochondria Activate Endothelial Cells (p 43)Download figureDownload PowerPointMitochondria released from activated monocytes are proinflammatory, report Puhm et al.Extracellular vesicles, such as microvesicles (MVs), are small, membrane-bound packages released from cells that enable cell-to-cell communication. While these vesicles can be released under normal conditions, their abundance, contents, and origin may alter with, and even influence, disease states. MVs released from monocytes after bacterial LPS stimulation, for example, have been shown to contain abundant mitochondrial proteins. Mitochondrial DNA-containing MVs have also been reported in a mouse model of inflammation. Puhm and colleagues, therefore, hypothesized that the mitochondrial content of MVs might actively contribute to proinflammatory effects. The team found that LPS-stimulated monocytes not only release mitochondria-containing MVs but also free mitochondria themselves. These free and MV-encapsulated mitochondria were shown to induce endothelial cells to produce the inflammatory cytokines TNFα and interferon. Circulating MVs collected from LPS-injected human volunteers also prompted such endothelial cytokine production. Furthermore, inhibition of mitochondrial activity drastically reduced this proinflammatory capacity of monocyte-released MVs. Together, the results indicate that released mitochondria, whether free or in MVs, may be key players in certain inflammatory diseases.Macrophage Smad3 in Myocardial Infarction (p 55)Download figureDownload PowerPointSmad3 in heart macrophages protects against excess infarct damage, say Chen et al.After an injury, increased local levels of cytokine TGF-β can, depending on conditions, stimulate macrophages to adopt either proinflammatory or anti-inflammatory phenotypes, both of which involve activation of signal transducer Smad3. Since inflammation can influence the outcome of injuries, including myocardial infarctions, Chen and colleagues investigated the overall impact of the TGF-β/Smad3 pathway in postinfarction macrophages. The team found that mice engineered to lack Smad3 in macrophages faired poorly following infarction. Survival was reduced compared with control mice and the animals’ hearts exhibited increased adverse remodeling and greater impairment of function. The Smad3-lacking cells themselves showed perturbed phagocytic activity, sustained expression of proinflammatory genes, and reduced production of anti-inflammatory mediators when compared with control macrophages. These results suggest Smad3 is necessary for postinfarction macrophages to transition to an anti-inflammatory, phagocytic phenotype that protects against excess remodeling. Smad3 inhibition in cardiomyocytes has been reported to be protective against infarction damage, however. So, any potential Smad3-modifying therapies would require cell-type–specific deployment, say the authors. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesRelationship Between Serum Alpha-Tocopherol and Overall and Cause-Specific MortalityJiaqi Huang, et al. Circulation Research. 2019;125:29-40Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial CellsFlorian Puhm, et al. Circulation Research. 2019;125:43-52Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating InflammationBijun Chen, et al. Circulation Research. 2019;125:55-70 June 21, 2019Vol 125, Issue 1 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000281 Originally publishedJune 20, 2019 PDF download Advertisement

Gene Therapy for Catecholaminergic Polymorphic Ventricular Tachycardia by Inhibition of Ca2+/Calmodulin-Dependent Kinase II.

Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models. We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide [AIP], is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes. AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2R176Q/+) effectively suppressed ventricular arrhythmias induced by either β-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ≈50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP. This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.

Ischemic N-Acetylneuraminic Acid Release Impairs Mitochondrial Hexokinases II and Autophagy in Cardiomyocytes

Background: Elevated levels of circulating N-acetylneuraminic acid (Neu5Ac) is considered as an independent risk factor for coronary artery diseases. This study aims to find the potential impact of free Neu5Ac on cardiomyocyte injury. Methods: The impact of free Neu5Ac on mitochondrial hexokinases II (HK-II) and autophagy was investigated in primary cardiomyocytes and the mouse heart subjected to hypoxic insult. Findings: Hypoxia activated neuraminidase-1(Neu-1) to induce sialic acid release in cardiomyocytes. Free Neu5Ac bound to HK-II and impaired mitochondrial function, leading to cell apoptosis. Nutrient deprivation elicits autophagy for cell salvage. HK-II binds and inactivates mTOR. However, Neu5Ac disrupted the binding and impaired autophagic response by reactivating mTOR, evidenced by impaired autophagic flux with increased p62 expression and reduced ratio of LC3-II/LC3-I. Impaired mitochondrial HK-II and autophagy were also observed in isoproterenol-induced ischemic heart of mice. By inhibiting neuraminidase, anti-influenza drugs oseltamivir and zanamivir reduced sialic acid release with improved mitochondrial HK-II and autophagy, resultantly rescuing cardiomyocytes survival from ischemic injury. Interpretation: Myocardial ischemia activates neuraminidase, and the released Neu5Ac acts as a mediator to impair cardiomyocytes. These findings establish a previously unrecognized role of Neu5Ac in cardiac ischemic injury and suggest the potential of neuraminidase inhibition in cardiomyocytes. Funding: This work was supported in part by the National Science Fund of China for Distinguished Young Scholars, Major Research Plan of the National Science Foundation of China, and National Natural Science Foundation of China. Declaration of Interest: The authors declare no conflicts of interest that pertain to this work. Ethical Approval: All animal experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the procedures were approved by the Animal Care and Use Committee of China Pharmaceutical University.

Saxagliptin but Not Sitagliptin Inhibits CaMKII and PKC via DPP9 Inhibition in Cardiomyocytes.

Some oral anti-hyperglycemic drugs, including gliptins that inhibit dipeptidyl peptidase 4 (DPP4), have been linked to the increased risk of heart failure (HF) in type-2 diabetic patients. While the cardiovascular safety trial, TECOS, revealed no link between sitagliptin and the risk of HF, a substantial 27% increase in the hospitalization for HF was observed in type-2 diabetic patients treated with saxagliptin within the SAVOR-TIMI 53 trial. A previous in vitro study revealed that saxagliptin impairs the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-phospholamban (PLB)-sarcoplasmic reticulum Ca2+-ATPase 2a axis and protein kinase C (PKC) activity in cardiomyocytes leading to impaired cardiac contractility and electrophysiological function. However, the link between saxagliptin and its target proteins (CaMKII and PKC) remains to be explored. Since DPP8 and DPP9 (but not DPP4) are expressed by cardiomyocytes and saxagliptin is internalized by cardiomyocytes, we investigated whether DPP8/9 contribute to saxagliptin-mediated inhibition of CaMKII and PKC activity. Structural analysis revealed that the DPP4-saxagliptin interaction motif (S630, Y547) for the cyanopyrrolidine group is conserved in DPP8 (S755, Y669) and DPP9 (S730, Y644). Conversely, F357 that facilitates binding of the anchor lock domain of sitagliptin in the S2 extensive subsite of DPP4 is not conserved in DPP8/9. In parallel, unlike saxagliptin, sitagliptin did not affect phosphorylation of CaMKII/PLB or activity of PKC in HL-1 cardiomyocytes. These findings were recapitulated by pharmacological inhibition (TC-E-5007, a DPP8/9 antagonist) and knock-down of DPP9 (but not DPP8). In primary mouse ventricular cardiomyocytes, saxagliptin (but not sitagliptin) impaired Ca2+ transient relaxation and prolonged action potential duration (APD). These results suggest that saxagliptin-DPP9 interaction impairs the CaMKII-PLB and PKC signaling in cardiomyocytes. We reveal a novel and potential role of DPP9 in cardiac signaling. The interaction of saxagliptin with DPP9 may represent an underlying mechanism for the link between saxagliptin and HF. Elucidation of saxagliptin-DPP9 interaction and downstream events may foster a better understanding of the role of gliptins as modulators of cardiac signaling.

ALDH2 protects against alcoholic cardiomyopathy through a mechanism involving the p38 MAPK/CREB pathway and local renin-angiotensin system inhibition in cardiomyocytes

BackgroundAngiotensin II (Ang II) in the local cardiac renin-angiotensin system (RAS) is closely associated with alcoholic cardiomyopathy (ACM). Inhibition of local cardiac RAS has great significance in the treatment of ACM. Although aldehyde dehydrogenase 2 (ALDH2) has been demonstrated to protect against ACM through detoxification of aldehydes, the precise mechanisms are largely unknown. In the present study, we determined whether ALDH2 improved cardiac damage by inhibiting the local RAS in ACM and investigated the related regulatory mechanisms. Methods and resultsAdult male mice were fed with 5% ethanol or a control diet for 2months, with or without the ALDH2 activator Alda-1. Heavy ethanol consumption induced cardiac damage, increased angiotensinogen (AGT) and Ang II and decreased myocardial ALDH2 activity in hearts. ALDH2 activation improved ethanol-induced cardiac damage and decreased AGT and Ang II in hearts. In vitro, ALDH2 activation or overexpression decreased AGT and Ang II in cultured cardiomyocytes treated with 400mmol/L ethanol for 24h. Furthermore, p38 MAP kinase (p38 MAPK)/cyclic adenosine monophosphate response element-binding protein (CREB) pathway activation by ethanol increased AGT and Ang II in cardiomyocytes. In addition, ALDH2 activation or overexpression inhibited the p38 MAPK/CREB pathway leading to decreased AGT and Ang II in cardiomyocytes. We also found that p38 MAPK activation effectively mitigated Alda-1-decreased AGT and Ang II, the effect of which was reversed by inhibition of CREB. ConclusionsALDH2 decreased AGT and Ang II in the local cardiac RAS via inhibiting the p38 MAPK/CREB pathway in ACM, thus improving ethanol-induced cardiac damage.