Human Induced Pluripotent Stem Cells Derived from a Cardiac Somatic Source: Insights for an In-Vitro Cardiomyocyte Platform.

Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.

Action potential contour contributes to species differences in repolarization response to ß-adrenergic stimulation.

Aim: Repolarization response to ß-adrenergic (ß-AR) stimulation differs between guinea-pig and canine myocytes and, within the latter, between myocardial layers. Correlative analysis suggests that this may be due to differences in action potential (AP) contour. Here we tested whether AP contour may set the response of current and of repolarization to ß-AR stimulation (10 nM isoproterenol, ISO). Methods and results: The responses of AP and current to ISO were measured under I-clamp and "AP-clamp" in guinea-pig (GP), dog epicardial (DEPI) and dog subendocardial (DENDO) myocytes. Dynamic-clamp (DC) was used to evaluate the impact of AP features on AP response to ISO. ISO prolonged AP duration (APD) in GP myocytes, did not affect it in DENDO and shortened it in DEPI ones. The current induced by ISO (IISO) sharply differed between GP and canine myocytes and, to a lesser extent, between DENDO and DEPI ones. Differences in IISO profile likely important in setting APD response (time-to-peak, time-to-reversal), were minimized when canine myocytes where clamped with GP AP-waveforms and vice versa. Introduction of a "notch" in GP AP (by DC) was alone insufficient to affect the APD response to ISO; nevertheless, when incorporated in a GP AP-waveform, the main "canine" AP features ("notch" and low plateau potential) caused IISO of GP myocytes to acquire canine features. Conclusion: Early repolarization contour and level of plateau potential contribute to species-specificity of IISO profile. Changes in AP contour, also when generated by modulation of ISO-insensitive currents, may be crucial in setting APD response to ß-AR stimulation.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage.

Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

AIM: Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na+ current (INa), nifedipine, a blocker of L-type Ca2+ current (ICaL), and E4031, a blocker of the rapid component of delayed rectifier K+ current (IKr). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K+ current (IKs). CONCLUSION: In hiPSC-derived cardiomyocytes of cardiac origin, INa, ICaL, IKr, and IKs were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic cell origins. Further studies are warranted to characterize electrophysiological properties of hiPSC-derived cardiomyocytes generated from CPCs.

Combined action potential- and dynamic-clamp for accurate computational modelling of the cardiac IKr current.

In the present work Action-Potential clamp (APC) and Dynamic clamp (DC) were used in combination in order to optimize the Luo-Rudy (LRd) mathematical formulation of the guinea-pig rapid delayed rectifier K(+) current (IKr), and to validate the optimized model. To this end, IKr model parameters were adjusted to fit the experimental E4031-sensitive current (IE4031) recorded under APC in guinea-pig myocytes. Currents generated by LRd model (ILRd) and the optimized one (IOpt) were then compared by testing their suitability to replace IE4031 under DC. Under APC, ILRd was significantly larger than IE4031 (mean current densities 0.51±0.01 vs 0.21±0.05pA/pF; p<0.001), mainly because of different rectification. IOpt mean density (0.17±0.01pA/pF) was similar to the IE4031 one (NS); moreover, IOpt accurately reproduced IE4031 distribution along the different AP phases. Models were then compared under DC by blocking native IKr (5µM E4031) and replacing it with ILRd or IOpt. Whereas injection of ILRd overshortened AP duration (APD90) (by 25% of its pre-block value), IOpt injection restored AP morphology and duration to overlap pre-block values. This study highlights the power of APC and DC for the identification of reliable formulations of ionic current models. An optimized model of IKr has been obtained which fully reversed E4031 effects on the AP. The model strongly diverged from the widely used Luo-Rudy formulation; this can be particularly relevant to the in silico analysis of AP prolongation caused by IKr blocking or alterations.

Targeting senescence induced by age or chemotherapy with a polyphenol-rich natural extract improves longevity and healthspan in mice.

Accumulating senescent cells within tissues contribute to the progression of aging and age-related diseases. Botanical extracts, rich in phytoconstituents, present a useful resource for discovering therapies that could target senescence and thus improve healthspan. Here, we show that daily oral administration of a standardized extract of Salvia haenkei (Haenkenium (HK)) extended lifespan and healthspan of naturally aged mice. HK treatment inhibited age-induced inflammation, fibrosis and senescence markers across several tissues, as well as increased muscle strength and fur thickness compared with age-matched controls. We also found that HK treatment reduced acutely induced senescence by the chemotherapeutic agent doxorubicin, using p16LUC reporter mice. We profiled the constituent components of HK by mass spectrometry, and identified luteolin-the most concentrated flavonoid in HK-as a senomorphic compound. Mechanistically, by performing surface plasmon resonance and in situ proximity ligation assay, we found that luteolin disrupted the p16-CDK6 interaction. This work demonstrates that administration of HK promotes longevity in mice, possibly by modulating cellular senescence and by disrupting the p16-CDK6 interaction.

Altered functional differentiation of mesoangioblasts in a genetic myopathy.

Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts (cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from ß-sarcoglycan-null mice (ßSG(-/-)), a murine model of genetic myopathy with early myocardial involvement. Although complementation with ßSG gene was inconsequential, knock-in of miRNA669a (missing in ßSG(-/-) cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of ßSG(-/-) cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes (CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca(2+)-handling properties of ßSG(-/-) cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca(2+)-spark properties and RyR isoform expression, distinguished ßSG(-/-) cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in ßSG(-/-) cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by ßSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.

A dynamic clamping approach using in silico IK1 current for discrimination of chamber-specific hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents.

Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging.

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (INaL) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca2+ leak. Electrical and intracellular Ca2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.

SERCA2a stimulation by istaroxime improves intracellular Ca2+ handling and diastolic dysfunction in a model of diabetic cardiomyopathy.

AIMS: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. METHODS AND RESULTS: Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. CONCLUSIONS: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart.

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit(+) CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit(+) CSCs.

Structural and Electrophysiological Changes in a Model of Cardiotoxicity Induced by Anthracycline Combined With Trastuzumab.

BACKGROUND: Combined treatment with anthracyclines (e.g., doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal growth factor receptor 2 (HER2; ErbB2) antibody, in patients with HER2-positive cancer is limited by cardiotoxicity, as manifested by contractile dysfunction and arrhythmia. The respective roles of the two agents in the cardiotoxicity of the combined therapy are incompletely understood. OBJECTIVE: To assess cardiac performance, T-tubule organization, electrophysiological changes and intracellular Ca2+ handling in cardiac myocytes (CMs) using an in vivo rat model of Dox/Trz-related cardiotoxicity. METHODS AND RESULTS: Adult rats received 6 doses of either Dox or Trz, or the two agents sequentially. Dox-mediated left ventricular (LV) dysfunction was aggravated by Trz administration. Dox treatment, but not Trz, induced T-tubule disarray. Moreover, Dox, but not Trz monotherapy, induced prolonged action potential duration (APD), increased incidence of delayed afterdepolarizations (DADs) and beat-to-beat variability of repolarization (BVR), and slower Ca2+ transient decay. Although APD, DADs, BVR and Ca2+ transient decay recovered over time after the cessation of Dox treatment, subsequent Trz administration exacerbated these abnormalities. Trz, but not Dox, reduced Ca2+ transient amplitude and SR Ca2+ content, although only Dox treatment was associated with SERCA downregulation. Finally, Dox treatment increased Ca2+ spark frequency, resting Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and long-lasting Ca2+ release events (so-called Ca2+ "embers"), partially reproduced by Trz treatment. CONCLUSION: These results suggest that in vivo Dox but not Trz administration causes T-tubule disarray and pronounced changes in electrical activity of CMs. While adaptive changes may account for normal AP shape and reduced DADs late after Dox administration, subsequent Trz administration interferes with such adaptive changes. Intracellular Ca2+ handling was differently affected by Dox and Trz treatment, leading to SR instability in both cases. These findings illustrate the specific roles of Dox and Trz, and their interactions in cardiotoxicity and arrhythmogenicity.

Role of somatic cell sources in the maturation degree of human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) are a unique source of human cardiomyocytes for cardiac disease modeling. Incomplete functional maturation remains a major limitation, however. One of the determinants of iPSC-CM maturation is somatic cell origin. We therefore compared iPSC-CMs derived from different somatic cell sources. METHODS: Cardiac-derived mesenchymal progenitor cells (CPCs), bone marrow-derived mesenchymal stem cells (BMCs), and human dermal fibroblasts (HDFs) from same patients were reprogrammed into iPSCs and differentiated into iPSC-CMs. Expression of cardiac-specific genes, caffeine-responsive cells, and electrophysiological properties of differentiated cells were analyzed. To assess the contribution of epigenetic memory toward differences in gene expression observed during cardiac differentiation, DNA methylation patterns were determined in the early mesodermal cardiac promoter NKX2-5 and KCNQ1, which encodes for the pore-forming α-subunit of the slow component of delayed-rectifier potassium current (IKs). RESULTS: Cardiac genes (MYH6, TNNI3, KCNQ1, KCNE1) were upregulated in CPC-vs. BMC- and HDF-iPSC-CMs. At early differentiation stages, CPC-iPSC-CMs displayed higher numbers of caffeine-responsive cells than BMC- and HDF-iPSC-CMs. The hERG1 (KV11.1) blocker, E4031, followed by the IKs blocker, JNJ303, increased extracellular field potential duration in CPC-iPSC-CMs to a greater extent than in BMC- and HDF-iPSC-CMs. The promoter region of NKX2-5 was more highly methylated in BMCs and HDFs compared to CPCs, and to a lesser extent in BMC-iPSCs compared to CPC-iPSCs. CONCLUSIONS: These results suggest that human iPSCs from cardiac somatic cell sources may display enhanced capacity toward cardiac re-differentiation compared to non-cardiac cell sources, and that epigenetic mechanisms may play a role in this regard.

Modulation of sarcoplasmic reticulum function by PST2744 [istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] in a pressure-overload heart failure model.

PST2744 [Istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] is a novel inotropic agent that enhances sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2 activity. We investigated the istaroxime effect on Ca(2+) handling abnormalities in myocardial hypertrophy/failure (HF). Guinea pig myocytes were studied 12 weeks after aortic banding (AoB) and compared with those of sham-operated animals (sham). The gain of calcium-induced Ca(2+) release (CICR), sarcoplasmic reticulum (SR) Ca(2+) content, Na(+)/Ca(2+) exchanger (NCX) function, and the rate of SR reloading after caffeine-induced depletion (SR Ca(2+) uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca(2+) and membrane currents. HF characterization: AoB caused hypertrophy and failure in 100 and 25% of animals, respectively. Although CICR gain during constant pacing was preserved, SR Ca(2+) content and SR Ca(2+) uptake were strongly depressed. Resting Ca(2+) and the slope of the Na(+)/Ca(2+) exchanger current (I(NCX))/Ca(2+) relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca(2+) content, and SR Ca(2+) uptake rate were increased by istaroxime in sham myocytes and, to a significantly larger extent, in AoB myocytes; this led to almost complete recovery of SR Ca(2+) uptake in AoB myocytes. Istaroxime increased resting Ca(2+) and the slope of the I(NCX)/Ca(2+) relationship similarly in sham and AoB myocytes. Istaroxime failed to increase SERCA activity in skeletal muscle microsomes devoid of phospholamban. Thus, clear-cut abnormalities in Ca(2+) handling occurred in this model of hypertrophy, with mild decompensation. Istaroxime enhanced SR function more in HF myocytes than in normal ones; almost complete drug-induced recovery suggests a purely functional nature of SR dysfunction in this HF model.

Cardioprotection by cardiac progenitor cell-secreted exosomes: role of pregnancy-associated plasma protein-A.

Aims: Cell therapy trials using cardiac-resident progenitor cells (CPCs) and bone marrow-derived mesenchymal stem/progenitor cells (BMCs) in patients after myocardial infarction have provided encouraging results. Exosomes, nanosized extracellular vesicles of endosomal origin, figure prominently in the bioactivities of these cells. However, a head-to-head comparison of exosomes from the two cell types has not been performed yet. Methods and results: CPCs and BMCs were derived from cardiac atrial appendage specimens and sternal bone marrow, respectively, from patients (n = 20; age, 69.9 ± 10.9) undergoing heart surgery for aortic valve disease and/or coronary artery disease. Vesicles were purified from cell conditioned media by centrifugation/filtration and ultracentrifugation. Vesicle preparations were predominantly composed of exosomes based on particle size and marker expression (CD9, CD63, CD81, Alix, and TSG-101). CPC-secreted exosomes prevented staurosporine-induced cardiomyocyte apoptosis more effectively than BMC-secreted exosomes. In vivo, CPC-secreted exosomes reduced scar size and improved ventricular function after permanent coronary occlusion in rats more efficiently than BMC-secreted exosomes. Both types of exosomes stimulated blood vessel formation. CPC-secreted exosomes, but not BMC-derived exosomes, enhanced ventricular function after ischaemia/reperfusion. Proteomics profiling identified pregnancy-associated plasma protein-A (PAPP-A) as one of the most highly enriched proteins in CPC vs. BMC exosomes. The active form of PAPP-A was detected on CPC exosome surfaces. These vesicles released insulin-like growth factor-1 (IGF-1) via proteolytic cleavage of IGF-binding protein-4 (IGFBP-4), resulting in IGF-1 receptor activation, intracellular Akt and ERK1/2 phosphorylation, decreased caspase activation, and reduced cardiomyocyte apoptosis. PAPP-A knockdown prevented CPC exosome-mediated cardioprotection both in vitro and in vivo. Conclusion: These results suggest that CPC-secreted exosomes may be more cardioprotective than BMC-secreted exosomes, and that PAPP-A-mediated IGF-1 release may explain the benefit. They illustrate a general mechanism whereby exosomes may function via an active protease on their surface, which releases a ligand in proximity to the transmembrane receptor bound by the ligand.

Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. METHODS AND RESULTS: Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. CONCLUSION: The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to ß-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

Direct inhibition of the pacemaker (If) current in rabbit sinoatrial node cells by genistein.

Genistein is a tyrosine kinase inhibitor which interferes with the activity of several ionic channels either by altering modulatory phosphorylating processes or by direct binding. In whole-cell conditions, genistein induces a partial inhibition of the pacemaker (I(f)) current recorded in cardiac sinoatrial and ventricular myocytes. We investigated the mechanism of action of genistein (50 microM) on the I(f) current in whole-cell, cell-attached, and inside-out configurations, and the measured fractional inhibitions were similar: 26.6, 27.2, and 33.6%, respectively. When ATP was removed from the whole-cell pipette solution no differences were revealed in the effect of the drug when compared to metabolically active cells. Genistein fully maintained its blocking ability even when herbimycin, a tyrosine kinase inhibitor, was added to the whole-cell ATP-free pipette solution. Genistein-induced block was independent of the gating state of the channel and did not display voltage or current dependence; this independence distinguishes genistein from all other f-channel blockers. When inside-out experiments were performed to test for a direct interaction with the channel, genistein, superfused on the intracellular side of the membrane, decreased the maximal I(f) conductance, and slightly shifted the current-activation curve to the left. Furthermore, the effect of genistein was independent of cAMP modulation. We conclude that, in addition to its tyrosine kinase-inhibitory properties, genistein also blocks I(f) by directly interacting with the channel, and thus cannot be considered a valuable pharmacological tool to investigate phosphorylation-dependent modulatory pathways of the I(f) current and of cardiac rhythm.

IKr Impact on Repolarization and Its Variability Assessed by Dynamic Clamp.

BACKGROUND: Repolarization and its stability are exquisitely sensitive to IKr features. Information on the relative importance of specific IKr abnormalities is missing and would assist in the evaluation of arrhythmogenic risk. METHODS AND RESULTS: In single guinea-pig myocytes, endogenous IKr was replaced by modeled IKr (mIKr) by dynamic clamp (DC) at a cycle length of 1 s. mIKr parameters were systematically modified, and the resulting changes in action potential duration (APD) and its short term variability (SD1) were measured. We observed that (1) IKr blockade increased SD1 more than expected by its dependency on APD; (2) mIKr completely reversed APD and SD1 changes caused by IKr blockade; (3) repolarization was most sensitive to inactivation shifts, which affected APD and SD1 concordantly; (4) activation shifts of the same magnitude had marginal impact on APD, but only when reducing mIKr, they significantly increased SD1; (5) changes in maximal conductance resulted in a pattern similar to that of activation shifts. CONCLUSIONS: The largest effect on repolarization and its stability are expected from changes in IKr inactivation. APD is less sensitive to changes in other IKr gating parameters, which are better revealed by SD1 changes. SD1 may be more sensitive than APD in detecting IKr-dependent repolarization abnormalities.

Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension.

AIMS: Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. METHODS AND RESULTS: PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca(2+) was increased and Ca(2+) release was facilitated. K(+) currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca(2+) content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. CONCLUSION: PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

Induced pluripotent stem cells: progress towards a biomedical application.

Some 5 years ago, Takahashi and Yamanaka first obtained induced pluripotent stem cells (iPSCs) by genetically 'reprogramming' adult somatic cells (fibroblasts). This breakthrough opened a new frontier in regenerative medicine, in which iPSCs might effectively replace embryonic stem cells (ESCs), with the additional advantage of permitting autologous transplant. Unfortunately, the risk of aberrant reprogramming and of the complications related to the use of transgenes in the process still hinders iPSC clinical application. Nevertheless, differentiation of iPSCs derived from patients may already provide a formidable platform for the in vitro analysis of human disease mechanisms and their modulation by drugs. Such an approach has already been validated by the finding that iPSCs obtained from patients with a specific genetic syndrome can be differentiated into cardiomyocytes retaining the gene abnormality and recapitulating, at the cell level, the syndrome functional phenotype. Unlike the use of iPSCs in regenerative therapy, their development as disease models is unencumbered by safety constraints. Whatever the intended use, the availability of reliable and reproducible methods for somatic cell reprogramming, iPSC expansion and differentiation is pivotal and remains a major challenge to date. The article by Burridge and coworkers describes a process encompassing all the phases in the preparation of precursor-derived cardiomyocytes, characterized by unprecedented efficiency and, most notably, applicable to different human precursors, including ESCs and iPSCs derived from multiple somatic cell types. Provided that the process will prove reproducible when applied by different laboratories, the contribution of Burridge and coworkers may represent a genuine leap in the development of precursor-derived technologies.