Otmar Pfister

CD31− but Not CD31+ Cardiac Side Population Cells Exhibit Functional Cardiomyogenic Differentiation

Heart failure remains a leading cause of morbidity and mortality. The cellular mechanism underlying the development of cardiac dysfunction is a decrease in the number of viable cardiomyocytes. Recent observations have suggested that the adult heart may contain a progenitor cell population. Side population (SP) cells, characterized by a distinct Hoechst dye efflux pattern, have been shown to exist in multiple tissues and are capable of tissue-specific differentiation. In this report, we confirm the existence of a cardiac SP cell population, immunophenotypically distinct from bone marrow SP cells. Moreover, we demonstrate that among cardiac SP cells, the greatest potential for cardiomyogenic differentiation is restricted to cells negative for CD31 expression and positive for stem cell antigen 1 (Sca1) expression (CD31−/Sca1+). Furthermore, we determine that CD31−/Sca1+ cardiac SP cells are capable of both biochemical and functional cardiomyogenic differentiation into mature cardiomyocytes, with expression of cardiomyocyte-specific transcription factors and contractile proteins, as well as stimulated cellular contraction and intracellular calcium transients indistinguishable from adult cardiomyocytes. We also determine the necessity of cell-extrinsic signaling through coupling, although not fusion, with adult cardiomyocytes in regulating cardiomyogenic differentiation of cardiac SP cells. We, therefore, conclude that CD31−/Sca1+ cardiac SP cells represent a distinct cardiac progenitor cell population, capable of cardiomyogenic differentiation into mature cardiomyocytes through a process mediated by cellular coupling with adult cardiomyocytes.

Restoration of Cardiac Progenitor Cells After Myocardial Infarction by Self-Proliferation and Selective Homing of Bone Marrow–Derived Stem Cells

Tissue-specific progenitor cells contribute to local cellular regeneration and maintain organ function. Recently, we have determined that cardiac side-population (CSP) cells represent a distinct cardiac progenitor cell population, capable of in vitro differentiation into functional cardiomyocytes. The response of endogenous CSP to myocardial injury, however, and the cellular mechanisms that maintain this cardiac progenitor cell pool in vivo remain unknown. In this report we demonstrate that local progenitor cell proliferation maintains CSP under physiologic conditions, with little contribution from extracardiac stem cell sources. Following myocardial infarction in adult mice, however, CSP cells are acutely depleted, both within the infarct and noninfarct areas. CSP pools are subsequently reconstituted to baseline levels within 7 days after myocardial infarction, through both proliferation of resident CSP cells, as well as through homing of bone marrow–derived stem cells (BMC) to specific areas of myocardial injury and immunophenotypic conversion of BMC to adopt a CSP phenotype. We, therefore, conclude that following myocardial injury, cardiac progenitor cell populations are acutely depleted and are reconstituted to normal levels by both self-proliferation and selective homing of BMC. Understanding and enhancing such processes hold enormous potential for therapeutic myocardial regeneration.

Kruppel-like factor 15 is a regulator of cardiomyocyte hypertrophy

Cardiac hypertrophy is a common response to injury and hemodynamic stress and an important harbinger of heart failure and death. Herein, we identify the Kruppel-like factor 15 (KLF15) as an inhibitor of cardiac hypertrophy. Myocardial expression of KLF15 is reduced in rodent models of hypertrophy and in biopsy samples from patients with pressure-overload induced by chronic valvular aortic stenosis. Overexpression of KLF15 in neonatal rat ventricular cardiomyocytes inhibits cell size, protein synthesis and hypertrophic gene expression. KLF15-null mice are viable but, in response to pressure overload, develop an eccentric form of cardiac hypertrophy characterized by increased heart weight, exaggerated expression of hypertrophic genes, left ventricular cavity dilatation with increased myocyte size, and reduced left ventricular systolic function. Mechanistically, a combination of promoter analyses and gel-shift studies suggest that KLF15 can inhibit GATA4 and myocyte enhancer factor 2 function. These studies identify KLF15 as part of a heretofore unrecognized pathway regulating the cardiac response to hemodynamic stress.

Role of the ATP-Binding Cassette Transporter Abcg2 in the Phenotype and Function of Cardiac Side Population Cells

Recently, the side population (SP) phenotype has been introduced as a reliable marker to identify subpopulations of cells with stem/progenitor cell properties in various tissues. We and others have identified SP cells from postmitotic tissues, including adult myocardium, in which they have been suggested to contribute to cellular regeneration following injury. SP cells are identified and characterized by a unique efflux of Hoechst 33342 dye. Abcg2 belongs to the ATP-binding cassette (ABC) transporter superfamily and constitutes the molecular basis for the dye efflux, hence the SP phenotype, in hematopoietic stem cells. Although Abcg2 is also expressed in cardiac SP (cSP) cells, its role in regulating the SP phenotype and function of cSP cells is unknown. Herein, we demonstrate that regulation of the SP phenotype in cSP cells occurs in a dynamic, age-dependent fashion, with Abcg2 as the molecular determinant of the cSP phenotype in the neonatal heart and another ABC transporter, Mdr1, as the main contributor to the SP phenotype in the adult heart. Using loss- and gain-of-function experiments, we find that Abcg2 tightly regulates cell fate and function. Adult cSP cells isolated from mice with genetic ablation of Abcg2 exhibit blunted proliferation capacity and augmented cell death. Conversely, overexpression of Abcg2 is sufficient to enhance cell proliferation, although with a limitation of cardiomyogenic differentiation. In summary, for the first time, we reveal a functional role for Abcg2 in modulating the proliferation, differentiation, and survival of adult cSP cells that goes beyond its distinct role in Hoechst dye efflux.

Redox-mediated reciprocal regulation of SERCA and Na+-Ca2+ exchanger contributes to sarcoplasmic reticulum Ca2+ depletion in cardiac myocytes.

Myocardial failure is associated with increased oxidative stress and abnormal excitation-contraction coupling characterized by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores and a reduction in Ca(2+)-transient amplitude. Little is known about the mechanisms whereby oxidative stress affects Ca(2+) handling and contractile function; however, reactive thiols may be involved. We used an in vitro cardiomyocyte system to test the hypothesis that short-term oxidative stress induces SR Ca(2+) depletion via redox-mediated regulation of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) and the sodium-Ca(2+) exchanger (NCX) and that this is associated with thiol oxidation. Adult rat ventricular myocytes paced at 5 Hz were superfused with H(2)O(2) (100 microM, 15 min). H(2)O(2) caused a progressive decrease in cell shortening followed by diastolic arrest, which was associated with decreases in SR Ca(2+) content, systolic [Ca(2+)](i), and Ca(2+)-transient amplitude, but no change in diastolic [Ca(2+)](i). H(2)O(2) caused reciprocal effects on the activities of SERCA (decreased) and NCX (increased). Pretreatment with the NCX inhibitor KB-R7943 before H(2)O(2) increased diastolic [Ca(2+)](i) and mimicked the effect of SERCA inhibition with thapsigargin. These functional effects were associated with oxidative modification of thiols on both SERCA and NCX. In conclusion, redox-mediated SR Ca(2+) depletion involves reciprocal regulation of SERCA and NCX, possibly via direct oxidative modification of both proteins.

Mesenchymal stem cells in the infarcted heart.

A loss of functional cardiomyocytes forms the cellular basis of cardiac dysfunction and heart failure. Stem cell based repletion of scarred myocardial tissue and regeneration of cardiomyocytes have been proposed as a potential treatment of ventricular dysfunction. In this review, we provide an overview of recent studies utilizing mesenchymal stem cells in cardiac regeneration and post-myocardial infarct therapy.

Regenerative therapy for cardiovascular disease

Recent insights into myocardial biology uncovered a hereto unknown regenerative capacity of the adult heart. The discovery of dividing cardiomyocytes and the identification and characterization of cardiac stem and progenitor cells with myogenic and angiogenic potential have generated new hopes that cardiac regeneration and repair might become a therapeutic option. During the past decade, multiple candidate cells have been proposed for cardiac regeneration, and their mechanisms of action in the myocardium have been explored. Initial clinical trials have focused on the use of bone marrow-derived cells to promote myocardial regeneration in ischemic heart disease and have yielded very mixed results, with no clear signs of clinically meaningful functional improvement. Although the efficiency of bona fide cardiomyocyte generation is generally low, stem cells delivered into the myocardium act mainly via paracrine mechanisms. More recent studies taking advantage of cardiac committed cells (eg, resident cardiac progenitor cells or primed cardiogenic mesenchymal stem cells) showed promising results in first clinical pilot trials. Also, transplantation of cardiomyogenic cells generated by induced pluripotent stem cells and genetic reprogramming of dividing nonmyocytes into cardiomyocytes may constitute attractive new regenerative approaches in cardiovascular medicine in the future. We discuss advantages and limitations of specific cell types proposed for cell-based therapy in cardiology and give an overview of the first clinical trials using this novel therapeutic approach in patients with cardiovascular disease.

Isolation of Resident Cardiac Progenitor Cells by Hoechst 33342 Staining

Cardiac resident stem/progenitor cells are critical to the cellular and functional integrity of the heart by maintaining myocardial cell homeostasis. Given their central role in myocardial biology, resident cardiac progenitor cells have become a major focus in cardiovascular research. Identification of putative cardiac progenitor cells within the myocardium is largely based on the presence or absence of specific cell surface markers. Additional purification strategies take advantage of the ability of stem cells to efficiently efflux vital dyes such as Hoechst 33342. During fluoresence activated cell sorting (FACS) such Hoechst-extruding cells appear to the side of Hoechst-dye retaining cells and have thus been termed side population (SP) cells. We have shown that cardiac SP cells that express stem cell antigen 1 (Sca-1) but not CD31 are cardiomyogenic, and thus represent a putative cardiac progenitor cell population. This chapter describes the methodology for the isolation of resident cardiac progenitor cells utilizing the SP phenotype combined with stem cell surface markers.

Cardiovascular Management of Cancer Patients With Chemotherapy-Associated Left Ventricular Systolic Dysfunction in Real-World Clinical Practice

BACKGROUND Chemotherapy-induced left ventricular systolic dysfunction (LVSD) may limit survival in cancer patients and therefore should be treated timely with appropriate heart failure medication. This study aimed to evaluate quality of cardiac care in cancer patients with documented chemotherapy-induced LVSD in real-world clinical practice. METHODS Using an institutional echo database, we screened 1,520 cancer patients for first documentation of chemotherapy-associated LVSD, defined as left ventricular ejection fraction (LVEF) ≤45%. Hospital charts of all 63 patients meeting inclusion criteria were reviewed regarding patient characteristics and frequency of heart failure medication prescription. RESULTS Patients were 61 (interquartile range [IQR], 50-70) years old, mostly symptomatic, and had an average LVEF of 34 ± 8%. Most patients received anthracyclines (73%) and/or alkylating agents (73%) as part of their chemotherapeutic regimen. Median time from cancer diagnosis to first documentation of LVSD was 2.2 (0.7-5.2) years. Fewer than two-thirds of patients received guideline-recommended heart failure medication, and only one-half of patients received cardiology consult. Cardiology consultation was associated with a significantly higher frequency of heart failure medication prescription (100% vs. 52% for angiotensin-converting enzyme inhibitor or angiotensin receptor blocker (P < .0001); 94% vs. 41% for beta-blocker (P < .0001) and better survival (71% vs. 41%; P < .05). CONCLUSIONS Chemotherapy-associated LVSD is insufficiently treated in cancer patients. Cardiology consultation improves rates of heart failure medication and therefore should be advocated in all patients with chemotherapy-induced LVSD.

Heart failure with mid-range ejection fraction: a distinct clinical entity? Insights from the Trial of Intensified versus standard Medical therapy in Elderly patients with Congestive Heart Failure (TIME-CHF)

While the conditions of heart failure (HF) with reduced (HFrEF, LVEF < 40%) and preserved (HFpEF, LVEF ≥ 50%) left ventricular ejection fraction (LVEF) are well characterized, it is unknown whether patients with HF and mid‐range LVEF (HFmrEF, LVEF 40–49%) have to be regarded as a separate clinical entity. The aim of this study was to characterize these three populations and to compare outcome and response to therapy.