Notch signaling modulates the electrical behavior of cardiomyocytes.

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K+ Kv currents, critical determinants of the electrical repolarization of myocytes.

p53 Modulates the Fate of Cardiac Progenitor Cells Ex Vivo and in the Diabetic Heart In Vivo.

p53 is an important modulator of stem cell fate, but its role in cardiac progenitor cells (CPCs) is unknown. Here, we tested the effects of a single extra-copy of p53 on the function of CPCs in the presence of oxidative stress mediated by doxorubicin in vitro and type-1 diabetes in vivo. CPCs were obtained from super-p53 transgenic mice (p53-tg), in which the additional allele is regulated in a manner similar to the endogenous protein. Old CPCs with increased p53 dosage showed a superior ability to sustain oxidative stress, repair DNA damage and restore cell division. With doxorubicin, a larger fraction of CPCs carrying an extra-copy of the p53 allele recruited γH2A.X reestablishing DNA integrity. Enhanced p53 expression resulted in a superior tolerance to oxidative stress in vivo by providing CPCs with defense mechanisms necessary to survive in the milieu of the diabetic heart; they engrafted in regions of tissue injury and in three days acquired the cardiomyocyte phenotype. The biological advantage provided by the increased dosage of p53 in CPCs suggests that this genetic strategy may be translated to humans to increase cellular engraftment and growth, critical determinants of successful cell therapy for the failing heart.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Hyperglycemia induces defective Ca2+ homeostasis in cardiomyocytes.

Diabetes and other metabolic conditions characterized by elevated blood glucose constitute important risk factors for cardiovascular disease. Hyperglycemia targets myocardial cells rendering ineffective mechanical properties of the heart, but cellular alterations dictating the progressive deterioration of cardiac function with metabolic disorders remain to be clarified. In the current study, we examined the effects of hyperglycemia on cardiac function and myocyte physiology by employing mice with high blood glucose induced by administration of streptozotocin, a compound toxic to insulin-producing ß-cells. We found that hyperglycemia initially delayed the electrical recovery of the heart, whereas cardiac function became defective only after ~2 mo with this condition and gradually worsened with time. Prolonged hyperglycemia was associated with increased chamber dilation, thinning of the left ventricle (LV), and myocyte loss. Cardiomyocytes from hyperglycemic mice exhibited defective Ca2+ transients before the appearance of LV systolic defects. Alterations in Ca2+ transients involved enhanced spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR), reduced cytoplasmic Ca2+ clearance, and declined SR Ca2+ load. These defects have important consequences on myocyte contraction, relaxation, and mechanisms of rate adaptation. Collectively, our data indicate that hyperglycemia alters intracellular Ca2+ homeostasis in cardiomyocytes, hindering contractile activity and contributing to the manifestation of the diabetic cardiomyopathy. NEW & NOTEWORTHY: We have investigated the effects of hyperglycemia on cardiomyocyte physiology and ventricular function. Our results indicate that defective Ca2+ handling is a critical component of the progressive deterioration of cardiac performance of the diabetic heart.

Reduction in Kv Current Enhances the Temporal Dispersion of the Action Potential in Diabetic Myocytes: Insights From a Novel Repolarization Algorithm.

BACKGROUND: Diabetes is associated with prolongation of the QT interval of the electrocardiogram and enhanced dispersion of ventricular repolarization, factors that, together with atherosclerosis and myocardial ischemia, may promote the occurrence of electrical disorders. Thus, we tested the possibility that alterations in transmembrane ionic currents reduce the repolarization reserve of myocytes, leading to action potential (AP) prolongation and enhanced beat-to-beat variability of repolarization. METHODS AND RESULTS: Diabetes was induced in mice with streptozotocin (STZ), and effects of hyperglycemia on electrical properties of whole heart and myocytes were studied with respect to an untreated control group (Ctrl) using electrocardiographic recordings in vivo, ex vivo perfused hearts, and single-cell patch-clamp analysis. Additionally, a newly developed algorithm was introduced to obtain detailed information of the impact of high glucose on AP profile. Compared to Ctrl, hyperglycemia in STZ-treated animals was coupled with prolongation of the QT interval, enhanced temporal dispersion of electrical recovery, and susceptibility to ventricular arrhythmias, defects observed, in part, in the Akita mutant mouse model of type I diabetes. AP was prolonged and beat-to-beat variability of repolarization was enhanced in diabetic myocytes, with respect to Ctrl cells. Density of Kv K(+) and L-type Ca(2+) currents were decreased in STZ myocytes, in comparison to cells from normoglycemic mice. Pharmacological reduction of Kv currents in Ctrl cells lengthened AP duration and increased temporal dispersion of repolarization, reiterating features identified in diabetic myocytes. CONCLUSIONS: Reductions in the repolarizing K(+) currents may contribute to electrical disturbances of the diabetic heart.

Myocyte repolarization modulates myocardial function in aging dogs.

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.

Rescue of neonatal cardiac dysfunction in mice by administration of cardiac progenitor cells in utero.

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family. We previously showed that disruption of the Speg gene locus in mice leads to a dilated cardiomyopathy with immature-appearing cardiomyocytes. Here we show that cardiomyopathy of Speg(-/-) mice arises as a consequence of defects in cardiac progenitor cell (CPC) function, and that neonatal cardiac dysfunction can be rescued by in utero injections of wild-type CPCs into Speg(-/-) foetal hearts. CPCs harvested from Speg(-/-) mice display defects in clone formation, growth and differentiation into cardiomyocytes in vitro, which are associated with cardiac dysfunction in vivo. In utero administration of wild-type CPCs into the hearts of Speg(-/-) mice results in CPC engraftment, differentiation and myocardial maturation, which rescues Speg(-/-) mice from neonatal heart failure and increases the number of live births by fivefold. We propose that in utero administration of CPCs may have future implications for treatment of neonatal heart diseases.

Late Na(+) current and protracted electrical recovery are critical determinants of the aging myopathy.

The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na(+) current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca(2+) cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca(2+) transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.

Aging Effects on Cardiac Progenitor Cell Physiology.

Cardiac aging has been confounded by the concept that the heart is a postmitotic organ characterized by a predetermined number of myocytes, which is established at birth and largely preserved throughout life until death of the organ and organism. Based on this premise, the age of cardiac cells should coincide with that of the organism; at any given time, the heart would be composed of a homogeneous population of myocytes of identical age. The discovery that stem cells reside in the heart and generate cardiac cell lineages has imposed a reconsideration of the mechanisms implicated in the manifestations of the aging myopathy. The progressive alterations of terminally differentiated myocytes, and vascular smooth muscle cells and endothelial cells may represent an epiphenomenon dictated by aging effects on cardiac progenitor cells (CPCs). Changes in the properties of CPCs with time may involve loss of self-renewing capacity, increased symmetric division with formation of daughter committed cells, partial depletion of the primitive pool, biased differentiation to the fibroblast fate, impaired ability to migrate, and forced entry into an irreversible quiescent state. Telomere shortening is a major variable of cellular senescence and organ aging, and support the notion that CPCs with critically shortened or dysfunctional telomeres contribute to myocardial aging and chronic heart failure. These defects constitute the critical variables that define the aging myopathy in humans. Importantly, a compartment of functionally competent human CPCs persists in the decompensated heart pointing to stem cell therapy as a novel form of treatment for the aging myopathy.

A Novel Class of Human Cardiac Stem Cells.

Following the recognition that hematopoietic stem cells improve the outcome of myocardial infarction in animal models, bone marrow mononuclear cells, CD34-positive cells, and mesenchymal stromal cells have been introduced clinically. The intracoronary or intramyocardial injection of these cell classes has been shown to be safe and to produce a modest but significant enhancement in systolic function. However, the identification of resident cardiac stem cells in the human heart (hCSCs) has created great expectation concerning the potential implementation of this category of autologous cells for the management of the human disease. Although phase 1 clinical trials have been conducted with encouraging results, the search for the most powerful hCSC for myocardial regeneration is in its infancy. This manuscript discusses the efforts performed in our laboratory to characterize the critical biological variables that define the growth reserve of hCSCs. Based on the theory of the immortal DNA template, we propose that stem cells retaining the old DNA represent 1 of the most powerful cells for myocardial regeneration. Similarly, the expression of insulin-like growth factor-1 receptors in hCSCs recognizes a cell phenotype with superior replicating reserve. However, the impressive recovery in ventricular hemodynamics and anatomy mediated by clonal hCSCs carrying the "mother" DNA underscores the clinical relevance of this hCSC class for the treatment of human heart failure.

Origin of cardiomyocytes in the adult heart.

This review article discusses the mechanisms of cardiomyogenesis in the adult heart. They include the re-entry of cardiomyocytes into the cell cycle; dedifferentiation of pre-existing cardiomyocytes, which assume an immature replicating cell phenotype; transdifferentiation of hematopoietic stem cells into cardiomyocytes; and cardiomyocytes derived from activation and lineage specification of resident cardiac stem cells. The recognition of the origin of cardiomyocytes is of critical importance for the development of strategies capable of enhancing the growth response of the myocardium; in fact, cell therapy for the decompensated heart has to be based on the acquisition of this fundamental biological knowledge.

Cardiac stem cell niches.

The critical role that stem cell niches have in cardiac homeostasis and myocardial repair following injury is the focus of this review. Cardiac niches represent specialized microdomains where the quiescent and activated state of resident stem cells is regulated. Alterations in niche function with aging and cardiac diseases result in abnormal sites of cardiomyogenesis and inadequate myocyte formation. The relevance of Notch1 signaling, gap-junction formation, HIF-1α and metabolic state in the regulation of stem cell growth and differentiation within the cardiac niches are discussed.

Dermatologic manifestations and neuropathic symptoms in women with Fabry disease.

Fabry disease (angiokeratoma corporis diffusum universale) is a rare, progressive, X-linked lysosomal storage disease. Deficiency of the α-galactosidase A (α-gal A) enzyme leads to accumulation of neutral glycosphingolipids within vascular endothelial lysosomes of various organs, including skin, kidneys, heart, and brain (1). We herein describe the case of a 30-year-old female presenting two classic signs of Fabry disease, angiokeratomas and episodic acroparesthesias, in the absence of other clinical manifestations. An haplotype corresponding to the combination of three different nucleotide polymorphic variants (g. 7192-7198del5+ g. 10115A>G + g. 10956 C>T) at the heterozygous state, was identified (2).

Cardiac stem cells: biology and clinical applications.

SIGNIFICANCE: Heart disease is the primary cause of death in the industrialized world. Cardiac failure is dictated by an uncompensated reduction in the number of viable and fully functional cardiomyocytes. While current pharmacological therapies alleviate the symptoms associated with cardiac deterioration, heart transplantation remains the only therapy for advanced heart failure. Therefore, there is a pressing need for novel therapeutic modalities. Cell-based therapies involving cardiac stem cells (CSCs) constitute a promising emerging approach for the replenishment of the lost tissue and the restoration of cardiac contractility. RECENT ADVANCES: CSCs reside in the adult heart and govern myocardial homeostasis and repair after injury by producing new cardiomyocytes and vascular structures. In the last decade, different classes of immature cells expressing distinct stem cell markers have been identified and characterized in terms of their growth properties, differentiation potential, and regenerative ability. Phase I clinical trials, employing autologous CSCs in patients with ischemic cardiomyopathy, are being completed with encouraging results. CRITICAL ISSUES: Accumulating evidence concerning the role of CSCs in heart regeneration imposes a reconsideration of the mechanisms of cardiac aging and the etiology of heart failure. Deciphering the molecular pathways that prevent activation of CSCs in their environment and understanding the processes that affect CSC survival and regenerative function with cardiac pathologies, commonly accompanied by alterations in redox conditions, are of great clinical importance. FUTURE DIRECTIONS: Further investigations of CSC biology may be translated into highly effective and novel therapeutic strategies aiming at the enhancement of the endogenous healing capacity of the diseased heart.