Aims PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. Methods and results Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. Conclusion PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF.

Properties of the funny current (If) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of If in human-induced pluripotent stem cell (iPSC)–derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that If density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. If displayed the voltage-dependent block by caesium and reversed (Erev) at − 22 mV, compatibly with the 3:1 K+/Na+ permeability ratio. Lowering [Na+]o (30 mM) shifted the Erev to − 39 mV without affecting conductance. Increasing [K+]o (30 mM) shifted the Erev to − 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of If. Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of If voltage-dependence (EC50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC50, 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an If with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human If-related sinus arrhythmias.

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miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up-or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC).We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166 + SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow-or fast-activating I f currents; miR1OE SAN-like cells instead have only fast-activating I f with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and I f alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1.In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

In the early phases of the COVID-19 pandemic, drug repurposing was widely used to identify compounds that could improve the prognosis of symptomatic patients infected by SARS-CoV-2. Hydroxychloroquine (HCQ) was one of the first drugs used to treat COVID-19 due to its supposed capacity of inhibiting SARS-CoV-2 infection and replication in vitro. While its efficacy is debated, HCQ has been associated with QT interval prolongation and potentially Torsades de Pointes, especially in patients predisposed to developing drug-induced Long QT Syndrome (LQTS) as silent carriers of variants associated with congenital LQTS. If confirmed, these effects represent a limitation to the at-home use of HCQ for COVID-19 infection as adequate ECG monitoring is challenging. We investigated the proarrhythmic profile of HCQ with Multi-Electrode Arrays after exposure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from two healthy donors, one asymptomatic and two symptomatic LQTS patients. We demonstrated that: I) HCQ induced a concentration-dependent Field Potential Duration (FPD) prolongation and halted the beating at high concentration due to the combined effect of HCQ on multiple ion currents. II) hiPSC-CMs from healthy or asymptomatic carriers tolerated higher concentrations of HCQ and showed lower susceptibility to HCQ-induced electrical abnormalities regardless of baseline FPD. These findings agree with the clinical safety records of HCQ and demonstrated that hiPSC-CMs potentially discriminates symptomatic vs. asymptomatic mutation carriers through pharmacological interventions. Disease-specific cohorts of hiPSC-CMs may be a valid preliminary addition to assess drug safety in vulnerable populations, offering rapid preclinical results with valuable translational relevance for precision medicine.