Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype.

BACKGROUND: Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. METHODS AND RESULTS: Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. CONCLUSION: Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.

Somatostatin and Its Receptors in Myocardial Ischemia/Reperfusion Injury and Cardioprotection.

Little is known about the role of the neuropeptide somatostatin (SST) in myocardial ischemia/reperfusion injury and cardioprotection. Here, we investigated the direct cardiocytoprotective effect of SST on ischemia/reperfusion injury in cardiomyocyte cultures, as well as the expression of SST and its receptors in pig and human heart tissues. SST induced a bell-shaped, concentration-dependent cardiocytoprotection in both adult rat primary cardiomyocytes and H9C2 cells subjected to simulated ischemia/reperfusion injury. Furthermore, in a translational porcine closed-chest acute myocardial infarction model, ischemic preconditioning increased plasma SST-like immunoreactivity. Interestingly, SST expression was detectable at the protein, but not at the mRNA level in the pig left ventricles. SSTR1 and SSTR2, but not the other SST receptors, were detectable at the mRNA level by PCR and sequencing in the pig left ventricle. Moreover, remote ischemic conditioning upregulated SSTR1 mRNA. Similarly, SST expression was also detectable in healthy human interventricular septum samples at the protein level. Furthermore, SST-like immunoreactivity decreased in interventricular septum samples of patients with ischemic cardiomyopathy. SSTR1, SSTR2, and SSTR5 but not SST and the other SST receptors were detectable at the mRNA level by sequencing in healthy human left ventricles. In addition, in healthy human left ventricle samples, SSTR1 and SSTR2 mRNAs were expressed especially in vascular endothelial and some other cell types as detected by RNA Scope® in situ hybridization. This is the first demonstration that SST exerts a direct cardiocytoprotective effect against simulated ischemia/reperfusion injury. Moreover, SST is expressed in the heart tissue at the peptide level; however, it is likely to be of sensory neural origin since its mRNA is not detectable. SSTR1 and SSTR2 might be involved in the cardioprotective action of SST, but other mechanisms cannot be excluded.

Decorin Protects Cardiac Myocytes against Simulated Ischemia/Reperfusion Injury.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.