Circulating miR-133a-3p defines a low-risk subphenotype in patients with heart failure and central sleep apnea: a decision tree machine learning approach.

BACKGROUND: Patients with heart failure with reduced ejection fraction (HFrEF) and central sleep apnea (CSA) are at a very high risk of fatal outcomes. OBJECTIVE: To test whether the circulating miRNome provides additional information for risk stratification on top of clinical predictors in patients with HFrEF and CSA. METHODS: The study included patients with HFrEF and CSA from the SERVE-HF trial. A three-step protocol was applied: microRNA (miRNA) screening (n = 20), technical validation (n = 60), and biological validation (n = 587). The primary outcome was either death from any cause, lifesaving cardiovascular intervention, or unplanned hospitalization for worsening of heart failure, whatever occurred first. MiRNA quantification was performed in plasma samples using miRNA sequencing and RT-qPCR. RESULTS: Circulating miR-133a-3p levels were inversely associated with the primary study outcome. Nonetheless, miR-133a-3p did not improve a previously established clinical prognostic model in terms of discrimination or reclassification. A customized regression tree model constructed using the Classification and Regression Tree (CART) algorithm identified eight patient subphenotypes with specific risk patterns based on clinical and molecular characteristics. MiR-133a-3p entered the regression tree defining the group at the lowest risk; patients with log(NT-proBNP) ≤ 6 pg/mL (miR-133a-3p levels above 1.5 arbitrary units). The overall predictive capacity of suffering the event was highly stable over the follow-up (from 0.735 to 0.767). CONCLUSIONS: The combination of clinical information, circulating miRNAs, and decision tree learning allows the identification of specific risk subphenotypes in patients with HFrEF and CSA.

AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes.

AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by in vivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in in vivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of in vivo cardiomyocyte transduction.

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity.

AIMS: Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. METHODS AND RESULTS: The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. CONCLUSION: Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

miR-486 attenuates cardiac ischemia/reperfusion injury and mediates the beneficial effect of exercise for myocardial protection.

Exercise and its regulated molecules have myocardial protective effects against cardiac ischemia/reperfusion (I/R) injury. The muscle-enriched miR-486 was previously identified to be upregulated in the exercised heart, which prompted us to investigate the functional roles of miR-486 in cardiac I/R injury and to further explore its potential in contributing to exercise-induced protection against I/R injury. Our data showed that miR-486 was significantly downregulated in the heart upon cardiac I/R injury. Both preventive and therapeutic interventions of adeno-associated virus 9 (AAV9)-mediated miR-486 overexpression could reduce cardiac I/R injury. Using AAV9 expressing miR-486 with a cTnT promoter, we further demonstrated that cardiac muscle cell-targeted miR-486 overexpression was also sufficient to protect against cardiac I/R injury. Consistently, miR-486 was downregulated in oxygen-glucose deprivation/reperfusion (OGDR)-stressed cardiomyocytes, while upregulating miR-486 inhibited cardiomyocyte apoptosis through PTEN and FoxO1 inhibition and AKT/mTOR activation. Finally, we observed that miR-486 was necessary for exercise-induced protection against cardiac I/R injury. In conclusion, miR-486 is protective against cardiac I/R injury and myocardial apoptosis through targeting of PTEN and FoxO1 and activation of the AKT/mTOR pathway, and mediates the beneficial effect of exercise for myocardial protection. Increasing miR-486 might be a promising therapeutic strategy for myocardial protection.

MicroRNAs targeting the SARS-CoV-2 entry receptor ACE2 in cardiomyocytes.

The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a public health emergency of international concern as more than 15 million cases were reported by 24th July 2020. Angiotensin-converting enzyme 2 (ACE2) is a COVID-19 entry receptor regulating host cell infection. A recent study reported that ACE2 is expressed in cardiomyocytes. In this study, we aimed to explore if there are microRNA (miRNA) molecules which target ACE2 and which may be exploited to regulate the SARS-CoV-2 receptor. Our data reveal that both Ace2 mRNA and Ace2 protein levels are inhibited by miR-200c in rat primary cardiomyocytes and importantly, in human iPSC-derived cardiomyocytes. We report the first miRNA candidate that can target ACE2 in cardiomyocytes and thus may be exploited as a preventive strategy to treat cardiovascular complications of COVID-19.

Circulating Long Noncoding RNA LIPCAR Predicts Heart Failure Outcomes in Patients Without Chronic Kidney Disease.

The plasma levels of long noncoding RNA LIPCAR are elevated in heart failure (HF) patients with reduced ejection fraction and associated with left ventricular remodeling and poor outcomes. We studied whether the presence of chronic kidney disease (CKD), as defined by an estimated glomerular filtration rate value <60mL/(min·1.73m2) modified the associations of plasma LIPCAR with left ventricular remodeling and outcomes in HF patients. Two hundred and thirty-four patients (mean age 74 [9.14] years, 50% male) were enrolled and followed for 4.73 (0.24-7.25) years. Plasma LIPCAR was detected by real-time quantitative polymerase chain reaction. LIPCAR was increased ( P=0.005) in patients compared with 17 age- and sex-matched controls, directly correlated with age ( P=0.001) and with the maximal early transmitral flow velocity to the mean peak early diastolic velocity of the mitral annulus displacement ratio ( P=0.001) and inversely correlated with estimated glomerular filtration rate ( P<0.001). LIPCAR was associated with hospitalization for HF, cardiovascular death, and a composite of hospitalization for HF or cardiovascular death ( P≤0.010), these associations being dependent of estimated glomerular filtration rate. The interactions between estimated glomerular filtration rate and LIPCAR with respect to these outcomes were statistically significant or of borderline significance ( P≤0.060). LIPCAR was increased in CKD patients compared with non-CKD patients ( P=0.021). LIPCAR was independently associated with hospitalization for HF ( P≤0.039) only in non-CKD patients, but its addition to traditional risk factors did not improve risk prediction in these patients. In conclusion, plasma LIPCAR prognosticates outcomes in elderly HF patients without CKD. Thus, there is an effect modification of CKD on the association of circulating LIPCAR with outcomes in HF patients.