HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

Predicting individual-specific cardiotoxicity responses induced by tyrosine kinase inhibitors.

Introduction: Tyrosine kinase inhibitor drugs (TKIs) are highly effective cancer drugs, yet many TKIs are associated with various forms of cardiotoxicity. The mechanisms underlying these drug-induced adverse events remain poorly understood. We studied mechanisms of TKI-induced cardiotoxicity by integrating several complementary approaches, including comprehensive transcriptomics, mechanistic mathematical modeling, and physiological assays in cultured human cardiac myocytes. Methods: Induced pluripotent stem cells (iPSCs) from two healthy donors were differentiated into cardiac myocytes (iPSC-CMs), and cells were treated with a panel of 26 FDA-approved TKIs. Drug-induced changes in gene expression were quantified using mRNA-seq, changes in gene expression were integrated into a mechanistic mathematical model of electrophysiology and contraction, and simulation results were used to predict physiological outcomes. Results: Experimental recordings of action potentials, intracellular calcium, and contraction in iPSC-CMs demonstrated that modeling predictions were accurate, with 81% of modeling predictions across the two cell lines confirmed experimentally. Surprisingly, simulations of how TKI-treated iPSC-CMs would respond to an additional arrhythmogenic insult, namely, hypokalemia, predicted dramatic differences between cell lines in how drugs affected arrhythmia susceptibility, and these predictions were confirmed experimentally. Computational analysis revealed that differences between cell lines in the upregulation or downregulation of particular ion channels could explain how TKI-treated cells responded differently to hypokalemia. Discussion: Overall, the study identifies transcriptional mechanisms underlying cardiotoxicity caused by TKIs, and illustrates a novel approach for integrating transcriptomics with mechanistic mathematical models to generate experimentally testable, individual-specific predictions of adverse event risk.

Potentiation of combined p19Arf and interferon-beta cancer gene therapy through its association with doxorubicin chemotherapy.

Balancing safety and efficacy is a major consideration for cancer treatments, especially when combining cancer immunotherapy with other treatment modalities such as chemotherapy. Approaches that induce immunogenic cell death (ICD) are expected to eliminate cancer cells by direct cell killing as well as activation of an antitumor immune response. We have developed a gene therapy approach based on p19Arf and interferon-ß gene transfer that, similar to conventional inducers of ICD, results in the release of DAMPS and immune activation. Here, aiming to potentiate this response, we explore whether association between our approach and treatment with doxorubicin (Dox), a known inducer of ICD, could further potentiate treatment efficacy without inducing cardiotoxicity, a critical side effect of Dox. Using central composite rotational design analysis, we show that cooperation between gene transfer and chemotherapy killed MCA205 and B16F10 cells and permitted the application of reduced viral and drug doses. The treatments also cooperated to induce elevated levels of ICD markers in MCA205, which correlated with improved efficacy of immunotherapy in vivo. Treatment of subcutaneous MCA205 tumors associating gene transfer and low dose (10 mg/kg) chemotherapy resulted in inhibition of tumor progression. Moreover, the reduced dose did not cause cardiotoxicity as compared to the therapeutic dose of Dox (20 mg/kg). The association of p19Arf/interferon-ß gene transfer and Dox chemotherapy potentiated antitumor response and minimized cardiotoxicity.

WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that lead to damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins six, eight, 18 and tumor necrosis factor-alpha (TNF-α) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro.

Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

In vitro heterogeneity of porcine adipose tissue-derived stem cells.

Tissue-specific adult stem cells (ASC) are heterogeneous and characterized by a mix of progenitor cells that produce cells at various stages of differentiation, and ultimately different terminally differentiated cells. Understanding the heterogeneity of ASCs may lead to the development of improved protocols of cell isolation and optimized cell therapy clinical protocols. Using a combination of enzymatic and explant culture protocols, we obtained pADSC population, which is composed by two distinct morphologies: fibroblast-like cells (FLCs) and endothelial-like cells (ELCs). Both cell sub-types efficiently formed colonies, expressed CD90+/CD105+/CD44+, and differentially expressed such markers such as Nestin, Vimentin, Fibronectin, Cytokeratin, Connexin 43, CD31, CD34 and CD146 as well as the pluripotent stem cell markers Oct-4, Nanog and Sox2. Mixed populations of pADSCs did not lose their multipotentiality and the cells were able to undergo osteogenic, chondrogenic, adipogenic and myogenic differentiation. Furthermore, the mixed population spontaneously formed capillary tube structures. Our findings suggest that different subpopulations can be isolated from adipose tissue and that the ADSCs need to be better evaluated using a wide panel of different markers related to cell differentiation, which is important for stem cell therapy and regenerative medicine, particularly for advanced stem cells therapies - products that are currently under investigation or even use.

Cardioprotection Conferred by Sitagliptin Is Associated with Reduced Cardiac Angiotensin II/Angiotensin-(1-7) Balance in Experimental Chronic Kidney Disease.

Dipeptidyl peptidase IV (DPPIV) inhibitors are antidiabetic agents that exert renoprotective actions independently of glucose lowering. Cardiac dysfunction is one of the main outcomes of chronic kidney disease (CKD); however, the effects of DPPIV inhibition on cardiac impairment during CKD progression remain elusive. This study investigated whether DPPIV inhibition mitigates cardiac dysfunction and remodeling in rats with a 5/6 renal ablation and evaluated if these effects are associated with changes in the cardiac renin-angiotensin system (RAS). To this end, male Wistar rats underwent a 5/6 nephrectomy (Nx) or sham operation, followed by an 8-week treatment period with the DPPIV inhibitor sitagliptin (IDPPIV) or vehicle. Nx rats had lower glomerular filtration rate, overt albuminuria and higher blood pressure compared to sham rats, whereas CKD progression was attenuated in Nx + IDPPIV rats. Additionally, Nx rats exhibited cardiac hypertrophy and fibrosis, which were associated with higher cardiac DPPIV activity and expression. The sitagliptin treatment prevented cardiac fibrosis and mitigated cardiac hypertrophy. The isovolumic relaxation time (IRVT) was higher in Nx than in sham rats, which was suggestive of CKD-associated-diastolic dysfunction. Sitagliptin significantly attenuated the increase in IRVT. Levels of angiotensin II (Ang II) in the heart tissue from Nx rats were higher while those of angiotensin-(1-7) Ang-(1-7) were lower than that in sham rats. This cardiac hormonal imbalance was completely prevented by sitagliptin. Collectively, these results suggest that DPPIV inhibition may delay the onset of cardiovascular impairment in CKD. Furthermore, these findings strengthen the hypothesis that a crosstalk between DPPIV and the renin-angiotensin system plays a role in the pathophysiology of cardiorenal syndromes.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation.

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

Allogeneic pASC transplantation in humanized pigs attenuates cardiac remodeling post-myocardial infarction.

Cell therapy repair strategies using adult mesenchymal stromal cells have shown promising evidence to prevent cardiac deterioration in rodents even in the absence of robust differentiation of the cells into cardiomyocytes. We tested whether increasing doses of porcine adipose-tissue derived mesenchymal stem cells (pASCs) increase cardiac tissue perfusion in pigs post-myocardial infarction (MI) receiving angiotensin-converting-enzyme inhibitor (ACE inhibitors) and Beta-blockers similarly to patients. Female pigs were subjected to MI induction by sponge permanent occlusion of left circumflex coronary artery (LCx) generating approximately 10% of injured LV area with minimum hemodynamic impact. We assessed tissue perfusion by real time myocardial perfusion echocardiography (RTMPE) using commercial microbubbles before and following pASCs treatment. Four weeks after the occlusion of the left circumflex artery, we transplanted placebo or pASCs (1, 2 and 4x106 cells/Kg BW) into the myocardium. The highest dose of pASCs increased myocardial vessel number and blood flow in the border (56% and 3.7-fold, respectively) and in the remote area (54% and 3.9-fold, respectively) while the non-perfused scar area decreased (up to 38%). We also found an increase of immature collagen fibers, although the increase in total tissue collagen and types I and III was similar in all groups. Our results provide evidence that pASCs-induced stimulation of tissue perfusion and accumulation of immature collagen fibers attenuates adverse remodeling post-MI beyond the normal beneficial effects associated with ACE inhibition and beta-blockade.

Dipeptidyl Peptidase IV Inhibition Exerts Renoprotective Effects in Rats with Established Heart Failure.

Circulating dipeptidyl peptidase IV (DPPIV) activity is associated with worse cardiovascular outcomes in humans and experimental heart failure (HF) models, suggesting that DPPIV may play a role in the pathophysiology of this syndrome. Renal dysfunction is one of the key features of HF, but it remains to be determined whether DPPIV inhibitors are capable of improving cardiorenal function after the onset of HF. Therefore, the present study aimed to test the hypothesis that DPPIV inhibition by vildagliptin improves renal water and salt handling and exerts anti-proteinuric effects in rats with established HF. To this end, male Wistar rats were subjected to left ventricle (LV) radiofrequency ablation or sham operation. Six weeks after surgery, radiofrequency-ablated rats who developed HF were randomly divided into two groups and treated for 4 weeks with vildagliptin (120 mg/kg/day) or vehicle by oral gavage. Echocardiography was performed before (pretreatment) and at the end of treatment (post-treatment) to evaluate cardiac function. The fractional area change (FAC) increased (34 ± 5 vs. 45 ± 3%, p < 0.05), and the isovolumic relaxation time decreased (33 ± 2 vs. 27 ± 1 ms; p < 0.05) in HF rats treated with vildagliptin (post-treatment vs. pretreatment). On the other hand, cardiac dysfunction deteriorated further in vehicle-treated HF rats. Renal function was impaired in vehicle-treated HF rats as evidenced by fluid retention, low glomerular filtration rate (GFR) and high levels of urinary protein excretion. Vildagliptin treatment restored urinary flow, GFR, urinary sodium and urinary protein excretion to sham levels. Restoration of renal function in HF rats by DPPIV inhibition was associated with increased active glucagon-like peptide-1 (GLP-1) serum concentration, reduced DPPIV activity and increased activity of protein kinase A in the renal cortex. Furthermore, the anti-proteinuric effect of vildagliptin treatment in rats with established HF was associated with upregulation of the apical proximal tubule endocytic receptor megalin and of the podocyte main slit diaphragm proteins nephrin and podocin. Collectively, these findings demonstrate that DPPIV inhibition exerts renoprotective effects and ameliorates cardiorenal function in rats with established HF. Long-term studies with DPPIV inhibitors are needed to ascertain whether these effects ultimately translate into improved clinical outcomes.

Efeito aditivo do transplante de células-troncos adultas sobre a perfusão cardíaca pós-infarto em porcos tratados com beta-bloqueador e inibidor da enzima conversora de angiotensiva / Additive effect of transplantation of adult stem cells post-infarction on the cardiac perfusion in pigs treated with beta-blocker and angiotensin-converting enzyme inhibitor

Os efeitos benéficos associados à injeção intramiocárdica de células-tronco adultas, obtidos em roedores, não tem sido reproduzidos de modo consistente em modelos animais de grande porte e seres humanos. Neste trabalho testamos a hipótese que o transplante de células-tronco mesenquimais derivadas do tecido adiposo de porcos (pASC) aumenta a perfusão tecidual cardíaca em animais infartados e humanizados pelo tratamento com um inibidor da enzima conversora de angiotensina (iECA) e um ?-bloqueador. Os animais foram submetidos a oclusão da artéria coronária circunflexa esquerda (ACX) e 4 semanas após o IM, 4 grupos foram randomizados para receber injeção intramiocárdica de pASC nas doses de 1, 2 ou 4x10 ...

The beneficial effects associated with intramyocardial injection of adult stem cells in rodents have not been consistently reproduced in larger animals and humans. We evaluated the dose of porcine adipose-tissue derived mesenchymal stem cells (pASC) to increase cardiac tissue perfusion in pigs treated with ace-inhibitors and ?-blockers to mimic human management post-MI. Animals were subjected to LCx occlusion and 4 weeks after MI blinded randomized in 4 groups to receive intramyocardial injection of pASC (1, 2 and 4x10 ...

Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway.

Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H(+)-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H(+)-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H(+)-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H(+)-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway.

Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics, senescence, karyotype and plasticity after long-term cryopreservation.

We and others have provided evidence that adipose tissue-derived mesenchymal stem cells (ASCs) can mitigate rat cardiac functional deterioration after myocardial ischemia, even though the mechanism of action or the relevance of these findings to human conditions remains elusive. In this regard, the porcine model is a key translational step, because it displays heart anatomic-physiological features that are similar to those found in the human heart. Towards this end, we wanted to establish the cultural characteristics of porcine ASCs (pASCs) with or without long-term cryostorage, considering that allogeneic transplantation may also be a future option. Compared to fresh pASCs, thawed cells displayed 90-95% viability and no changes in morphological characteristics or in the expression of surface markers (being pASCs characterized by positive markers CD29(+); CD90(+); CD44(+); CD140b(+); CD105(+); and negative markers CD31(-); CD34(-); CD45(-) and SLA-DR(-); n = 3). Mean population doubling time was also comparable (64.26±15.11 hours to thawed cells vs. 62.74±18.07 hours to fresh cells) and cumulative population doubling increased constantly until Passage 10 (P10) in the entire cell population, with a small and gradual increase in senescence (P5, 3.25%±0.26 vs. 3.47%±0.32 and P10, 9.6%±0.29 vs. 10.67%±1.25, thawed vs. fresh; SA-ß-Gal staining). Chromosomal aberrations were not observed. In addition, under both conditions pASCs responded to adipogenic and osteogenic chemical cues in vitro. In conclusion, we have demonstrated the growth characteristics, senescence, and the capacity of pASCs to respond to chemical cues in vitro and have provided evidence that these properties are not influenced by cryostorage in 10% DMSO solution.

Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule.

We previously demonstrated that uroguanylin (UGN) significantly inhibits Na(+)/H(+) exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK(1)). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H(+) secretion in rat renal proximal segments, through a H(+)-sensitive microelectrode. UGN (1 µM) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK(1) cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK(1) cells. Finally, treatment of LLC-PK(1) cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides are intricately involved in the maintenance of salt and water homeostasis.