Alessandra Rossini

Human cardiac and bone marrow stromal cells exhibit distinctive properties related to their origin

AIMS Bone marrow mesenchymal stromal cell (BMStC) transplantation into the infarcted heart improves left ventricular function and cardiac remodelling. However, it has been suggested that tissue-specific cells may be better for cardiac repair than cells from other sources. The objective of the present work has been the comparison of in vitro and in vivo properties of adult human cardiac stromal cells (CStC) to those of syngeneic BMStC. METHODS AND RESULTS Although CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC. CONCLUSION Our findings demonstrate that although CStC and BMStC share a common stromal phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.

Functional diversity of electrogenic Na+–HCO3− cotransport in ventricular myocytes from rat, rabbit and guinea pig

The Na+–HCO3− cotransporter (NBC) is an important sarcolemmal acid extruder in cardiac muscle. The characteristics of NBC expressed functionally in heart are controversial, with reports suggesting electroneutral (NBCn; 1HCO3− : 1Na+; coupling coefficient N= 1) or electrogenic forms of the transporter (NBCe; equivalent to 2HCO3− : 1Na+; N= 2). We have used voltage‐clamp and epifluorescence techniques to compare NBC activity in isolated ventricular myocytes from rabbit, rat and guinea pig. Depolarization (by voltage clamp or hyperkalaemia) reversibly increased steady‐state pHi while hyperpolarization decreased it, effects seen only in CO2/HCO3−‐buffered solutions, and blocked by S0859 (cardiac NBC inhibitor). Species differences in amplitude of these pHi changes were rat > guinea pig ≈ rabbit. Tonic depolarization (−140 mV to −0 mV) accelerated NBC‐mediated pHi recovery from an intracellular acid load. At 0 mV, NBC‐mediated outward current at resting pHi was +0.52 ± 0.05 pA pF−1 (rat, n= 5), +0.26 ± 0.05 pA pF−1 (guinea pig, n= 5) and +0.10 ± 0.03 pA pF−1 (rabbit, n= 9), with reversal potentials near −100 mV, consistent with N= 2. The above results indicate a functionally active voltage‐sensitive NBCe in these species. Voltage‐clamp hyperpolarization negative to the reversal potential for NBCe failed, however, to terminate or reverse NBC‐mediated pHi‐recovery from an acid load although it was slowed significantly, suggesting electroneutral NBC may also be operational. NBC‐mediated pHi recovery was associated with a rise of [Na+]i at a rate ∼25% of that mediated via NHE, and consistent with an apparent NBC stoichiometry between N= 1 and N= 2. In conclusion, NBCe in the ventricular myocyte displays considerable functional variation among the three species tested (greatest in rat, least in rabbit) and may coexist with some NBCn activity.

Modelling intracellular H+ ion diffusion

Intracellular pH, an important modulator of cell function, is regulated by plasmalemmal proteins that transport H(+), or its equivalent, into or out of the cell. The pH(i) is also stabilised by high-capacity, intrinsic buffering on cytoplasmic proteins, oligopeptides and other solutes, and by the extrinsic CO(2)/HCO(3)(-) (carbonic) buffer. As mobility of these buffers is lower than for the H(+) ion, they restrict proton diffusion. In this paper we use computational approaches, based on the finite difference and finite element methods (FDM and FEM, respectively), for analysing the spatio-temporal behaviour of [H(+)] when it is locally perturbed. We analyse experimental data obtained for various cell-types (cardiac myocytes, duodenal enterocytes, molluscan neurons) where pH(i) has been imaged confocally using intracellular pH-sensitive dyes. We design mathematical algorithms to generate solutions for two-dimensional diffusion that fit data in terms of an apparent intracellular H(+) diffusion coefficient, D(H)(app). The models are used to explore how the spatial distribution of [H(+)](i) is affected by membrane H(+)-equivalent transport and by cell geometry. We then develop a mechanistic model, describing spatio-temporal changes of [H(+)](i) in a cardiac ventricular myocyte in terms of H(+)-shuttling on mobile buffers and H(+)-anchoring on fixed buffers. We also discuss how modelling may include the effects of extrinsic carbonic-buffering. Overall, our computational approach provides a framework for future analyses of the physiological consequences of pH(i) non-uniformity.

The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

pH‐Regulated Na+ Influx into the Mammalian Ventricular Myocyte: The Relative Role of Na+‐H+ Exchange and Na+‐HCO3− Co‐Transport

In the heart, intracellular Na+ concentration (Na+i) is a controller of intracellular Ca2+ signaling, and hence of key aspects of cell contractility and rhythm. Na+i will be influenced by variation in Na+ influx. In the present work, we consider one source of Na+ influx, sarcolemmal acid extrusion. Acid extrusion is accomplished by sarcolemmal H+ and HCO3− transporters that import Na+ ions while exporting H+ or importing HCO3−. The capacity of this system to import Na+ is enormous, up to four times the maximum capacity of the Na+‐K+ ATPase to extrude Na+ ions from the cell. In this review we consider the role of Na+‐H+ exchange (NHE) and Na+‐HCO3−co‐transport (NBC) in mediating Na+ influx into cardiac myocytes. We consider, in particular, the role of NBC, as so little is known about Na+ influx through this transporter. We show that both proteins mediate significant Na+ influx and that although, in the ventricular myocyte, NBC‐mediated Na+ influx is less than through NHE, the proportions may be altered under a variety of conditions, including exposure to catecholamines, membrane depolarization, and interference with activity of the enzyme, carbonic anhydrase.

H+ Ion Activation and Inactivation of the Ventricular Gap Junction. A Basis for Spatial Regulation of Intracellular pH

H+ ions are powerful modulators of cardiac function, liberated during metabolic activity. Among their physiological effects is a chemical gating of cell-to-cell communication, caused by H+-mediated closure of connexin (Cx) channels at gap junctions. This protects surrounding tissue from the damaging effects of local intracellular acidosis. Cx proteins (largely Cx-43 in ventricle) form multimeric pores between cells, permitting translocation of ions and other solutes up to ≈1 kDa. The channels are essential for electrical and metabolic coordination of a tissue. Here we demonstrate that, contrary to expectation, H+ ions can induce an increase of gap-junctional permeability. This occurs during modest intracellular acid loads in myocyte pairs isolated from mammalian ventricle. We show that the increase in permeability allows a local rise of [H+]i to dissipate into neighboring myocytes, thereby providing a mechanism for spatially regulating intracellular pH (pHi). During larger acid loads, the increased permeability is overridden by a more familiar H+-dependent inhibition (H+ inactivation). This restricts cell-to-cell H+ movement, while allowing sarcolemmal H+ transporters such as Na+/H+ exchange, to extrude the acid from the cell. The H+ sensitivity of Cx channels therefore defines whether junctional or sarcolemmal mechanisms are selected locally for the removal of an acid load. The bell-shaped pH dependence of permeability suggests that, in addition to H+ inactivation, an H+ activation process regulates the ensemble of Cx channels open at the junction. As well as promoting spatial pHi regulation, H+ activation of junctional permeability may link increased metabolic activity to improved myocardial coupling, the better to meet mechanical demand.

Proton Permeation Through the Myocardial Gap Junction

Abstract— Although protons can directly or indirectly gate solute permeability of the myocardial gap junction, there is little information regarding their own permeation, despite their importance in the regulation of myocardial contractility and rhythm. By pipette-loading of acid into guinea pig isolated, ventricular myocyte pairs while imaging pHi confocally using SNARF fluorescence, we have observed that protons permeate the junctional region. Permeation is inhibited by glycyrrhetinic acid, an agent that also increases intercellular electrical resistance, suggesting H+ permeation via gap junctions. The rate of spread of acid between cells appears to be limited by junctional permeation rather than by cytoplasmic diffusion. Mathematical analyses, combined with experiments using SNARF as a proton carrier, suggest that gap junctional H+ transmission may be accomplished physiologically by the permeation of intrinsic “proton-porter” molecules. We propose that proton flux through gap junctions will contribute to the dissipation of regional acid loads within the myocardium. This represents a mechanism for the local control of myocardial pHi.

Resident Cardiac Stem Cells

The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.

In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors

Adult human cardiac mesenchymal-like stromal cells (CStC) represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS) in the presence of 5 µM all-trans Retinoic Acid (ATRA), 5 µM Phenyl Butyrate (PB), and 200 µM diethylenetriamine/nitric oxide (DETA/NO), to create a novel epigenetically active cocktail (EpiC). Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker If current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy

Fibro-adipose substitution has a double detrimental effect on the myocardium in arrhythmogenic cardiomyopathy (ACM), worsening arrhythmogenesis by creating a non-conductive substrate, and causing ventricular dysfunction leading to heart failure. Notably, to-date no etiological therapy is available. This work introduces, for the first time, the stromal cardiac compartment as a key player in ACM ventricular adipose substitution: we demonstrated that cardiac human mesenchymal stromal cells undergo adipogenic differentiation both in ACM explanted hearts and in culture through a PKP2-dependent mechanism. Cardiac mesenchymal stromal cells constitute a suitable cellular platform for future mechanistic studies and a potential target for future therapies.