Sadia Mohsin

GDF11 Does Not Rescue Aging-Related Pathological Hypertrophy

RATIONALE Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. OBJECTIVE To determine whether and by what mechanism GDF11 rescues aging dependent PCH. METHODS AND RESULTS Twenty-four-month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. CONCLUSIONS Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.

Hrd1 and ER-Associated Protein Degradation, ERAD, Are Critical Elements of the Adaptive ER Stress Response in Cardiac Myocytes

RATIONALE Hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (Hrd1) is an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase that has been studied in yeast, where it contributes to ER protein quality control by ER-associated degradation (ERAD) of misfolded proteins that accumulate during ER stress. Neither Hrd1 nor ERAD has been studied in the heart, or in cardiac myocytes, where protein quality control is critical for proper heart function. OBJECTIVE The objective of this study were to elucidate roles for Hrd1 in ER stress, ERAD, and viability in cultured cardiac myocytes and in the mouse heart, in vivo. METHODS AND RESULTS The effects of small interfering RNA-mediated Hrd1 knockdown were examined in cultured neonatal rat ventricular myocytes. The effects of adeno-associated virus-mediated Hrd1 knockdown and overexpression were examined in the hearts of mice subjected to pressure overload-induced pathological cardiac hypertrophy, which challenges protein-folding capacity. In cardiac myocytes, the ER stressors, thapsigargin and tunicamycin increased ERAD, as well as adaptive ER stress proteins, and minimally affected cell death. However, when Hrd1 was knocked down, thapsigargin and tunicamycin dramatically decreased ERAD, while increasing maladaptive ER stress proteins and cell death. In vivo, Hrd1 knockdown exacerbated cardiac dysfunction and increased apoptosis and cardiac hypertrophy, whereas Hrd1 overexpression preserved cardiac function and decreased apoptosis and attenuated cardiac hypertrophy in the hearts of mice subjected to pressure overload. CONCLUSIONS Hrd1 and ERAD are essential components of the adaptive ER stress response in cardiac myocytes. Hrd1 contributes to preserving heart structure and function in a mouse model of pathological cardiac hypertrophy.

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy

Rationale: The relative actions and synergism between distinct myocardial-derived stem cell populations remain obscure. Ongoing debates on optimal cell population(s) for treatment of heart failure prompted implementation of a protocol for isolation of multiple stem cell populations from a single myocardial tissue sample to develop new insights for achieving myocardial regeneration. Objective: Establish a robust cardiac stem cell isolation and culture protocol to consistently generate 3 distinct stem cell populations from a single human heart biopsy. Methods and Results: Isolation of 3 endogenous cardiac stem cell populations was performed from human heart samples routinely discarded during implantation of a left ventricular assist device. Tissue explants were mechanically minced into 1 mm3 pieces to minimize time exposure to collagenase digestion and preserve cell viability. Centrifugation removes large cardiomyocytes and tissue debris producing a single cell suspension that is sorted using magnetic-activated cell sorting technology. Initial sorting is based on tyrosine-protein kinase Kit (c-Kit) expression that enriches for 2 c-Kit+ cell populations yielding a mixture of cardiac progenitor cells and endothelial progenitor cells. Flowthrough c-Kit− mesenchymal stem cells are positively selected by surface expression of markers CD90 and CD105. After 1 week of culture, the c-Kit+ population is further enriched by selection for a CD133+ endothelial progenitor cell population. Persistence of respective cell surface markers in vitro is confirmed both by flow cytometry and immunocytochemistry. Conclusions: Three distinct cardiac cell populations with individualized phenotypic properties consistent with cardiac progenitor cells, endothelial progenitor cells, and mesenchymal stem cells can be successfully concurrently isolated and expanded from a single tissue sample derived from human heart failure patients.

Unique Features of Cortical Bone Stem Cells Associated with Repair of the Injured Heart

RATIONALE Adoptive transfer of multiple stem cell types has only had modest effects on the structure and function of failing human hearts. Despite increasing the use of stem cell therapies, consensus on the optimal stem cell type is not adequately defined. The modest cardiac repair and functional improvement in patients with cardiac disease warrants identification of a novel stem cell population that possesses properties that induce a more substantial improvement in patients with heart failure. OBJECTIVE To characterize and compare surface marker expression, proliferation, survival, migration, and differentiation capacity of cortical bone stem cells (CBSCs) relative to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs), which have already been tested in early stage clinical trials. METHODS AND RESULTS CBSCs, MSCs, and CDCs were isolated from Gottingen miniswine or transgenic C57/BL6 mice expressing enhanced green fluorescent protein and were expanded in vitro. CBSCs possess a unique surface marker profile, including high expression of CD61 and integrin β4 versus CDCs and MSCs. In addition, CBSCs were morphologically distinct and showed enhanced proliferation capacity versus CDCs and MSCs. CBSCs had significantly better survival after exposure to an apoptotic stimuli when compared with MSCs. ATP and histamine induced a transient increase of intracellular Ca(2+) concentration in CBSCs versus CDCs and MSCs, which either respond to ATP or histamine only further documenting the differences between the 3 cell types. CONCLUSIONS CBSCs are unique from CDCs and MSCs and possess enhanced proliferative, survival, and lineage commitment capacity that could account for the enhanced protective effects after cardiac injury.

Role of STIM1 in Hypertrophy-Related Contractile Dysfunction

Rationale: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca2+ occurs in pathological hypertrophy and is central to myocyte remodeling. STIM1 (stromal interaction molecule 1) regulates Ca2+ signaling in many cell types by sensing low endoplasmic reticular Ca2+ levels and then coupling to plasma membrane Orai channels to induce a Ca2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy, but its role in electrical and mechanical phenotypic alterations is not well understood. Objective: To define the contributions of STIM1-mediated Ca2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine whether Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Methods and Results: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca2+-handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused a reduction of AP duration and reduced diastolic Ca2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca2+ stored within the sarcoplasmic reticulum and activated Ca2+/calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by coexpression of a dominant negative Orai construct. Conclusions: STIM1 can associate with Orai in cardiac myocytes to produce a Ca2+ influx pathway that can prolong the AP duration and load the sarcoplasmic reticulum and likely contributes to the altered electromechanical properties of the hypertrophied heart.Rationale: Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca 2+ occurs in pathological hypertrophy and is central to myocyte remodeling. Stromal Interaction Molecule 1 (STIM1) regulates Ca 2+ signaling in many cell types by sensing low endoplasmic reticular Ca 2+ levels and then coupling to plasma membrane Orai channels to induce a Ca 2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy but its role in electrical and mechanical phenotypic alterations are not well understood. Objective: To define the contributions of STIM1-mediated Ca 2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine if Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Methods and Results: Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca 2+ handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused in a reduction of AP duration and reduced diastolic Ca 2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca 2+ stored within the sarcoplasmic reticulum and activated Ca 2+ /calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by co-expression of a dominant negative Orai construct. Conclusions: STIM1 can associate with Orai in cardiac myocytes to produce a Ca 2+ influx pathway that can prolong the AP duration and load the SR and likely contributes to the altered electromechanical properties of the hypertrophied heart.

A Feline HFpEF Model with Pulmonary Hypertension and Compromised Pulmonary Function

Heart Failure with preserved Ejection Fraction (HFpEF) represents a major public health problem. The causative mechanisms are multifactorial and there are no effective treatments for HFpEF, partially attributable to the lack of well-established HFpEF animal models. We established a feline HFpEF model induced by slow-progressive pressure overload. Male domestic short hair cats (n = 20), underwent either sham procedures (n = 8) or aortic constriction (n = 12) with a customized pre-shaped band. Pulmonary function, gas exchange, and invasive hemodynamics were measured at 4-months post-banding. In banded cats, echocardiography at 4-months revealed concentric left ventricular (LV) hypertrophy, left atrial (LA) enlargement and dysfunction, and LV diastolic dysfunction with preserved systolic function, which subsequently led to elevated LV end-diastolic pressures and pulmonary hypertension. Furthermore, LV diastolic dysfunction was associated with increased LV fibrosis, cardiomyocyte hypertrophy, elevated NT-proBNP plasma levels, fluid and protein loss in pulmonary interstitium, impaired lung expansion, and alveolar-capillary membrane thickening. We report for the first time in HFpEF perivascular fluid cuff formation around extra-alveolar vessels with decreased respiratory compliance. Ultimately, these cardiopulmonary abnormalities resulted in impaired oxygenation. Our findings support the idea that this model can be used for testing novel therapeutic strategies to treat the ever growing HFpEF population.

Remodeling of repolarization and arrhythmia susceptibility in a myosin-binding protein C knockout mouse model

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases and among the leading causes of sudden cardiac death (SCD) in the young. The cellular mechanisms leading to SCD in HCM are not well known. Prolongation of the action potential (AP) duration (APD) is a common feature predisposing hypertrophied hearts to SCD. Previous studies have explored the roles of inward Na+ and Ca2+ in the development of HCM, but the role of repolarizing K+ currents has not been defined. The objective of this study was to characterize the arrhythmogenic phenotype and cellular electrophysiological properties of mice with HCM, induced by myosin-binding protein C (MyBPC) knockout (KO), and to test the hypothesis that remodeling of repolarizing K+ currents causes APD prolongation in MyBPC KO myocytes. We demonstrated that MyBPC KO mice developed severe hypertrophy and cardiac dysfunction compared with wild-type (WT) control mice. Telemetric electrocardiographic recordings of awake mice revealed prolongation of the corrected QT interval in the KO compared with WT control mice, with overt ventricular arrhythmias. Whole cell current- and voltage-clamp experiments comparing KO with WT mice demonstrated ventricular myocyte hypertrophy, AP prolongation, and decreased repolarizing K+ currents. Quantitative RT-PCR analysis revealed decreased mRNA levels of several key K+ channel subunits. In conclusion, decrease in repolarizing K+ currents in MyBPC KO ventricular myocytes contributes to AP and corrected QT interval prolongation and could account for the arrhythmia susceptibility.NEW & NOTEWORTHY Ventricular myocytes isolated from the myosin-binding protein C knockout hypertrophic cardiomyopathy mouse model demonstrate decreased repolarizing K+ currents and action potential and QT interval prolongation, linking cellular repolarization abnormalities with arrhythmia susceptibility and the risk for sudden cardiac death in hypertrophic cardiomyopathy.

Serum from CCl4-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis

Abstract Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4 injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4 which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson’s trichome staining was observed.

Stem Cells and Cardiac Repair

Discovery of tissue specific stem cells capable of forming cardiac cell types has revolutionized cardiac medicine. Not long ago, cardiac tissue regeneration was considered an impossible task. The last ten years however has seen an explosion of cell based therapeutic approaches stimulating cardiac regeneration and in the process augmenting function in the heart following injury. Encouraging preclinical results have paved the way for clinical applications of cell therapy and preliminary results obtained from various clinical trials indicate that stem cell transplantation increases cardiac function comparable to the existing interventions for treatment of heart diseases. These are promising outcomes that indicate that the cells have the ability to modulate cardiac repair programs leading to replacement of the lost tissue. Nevertheless, the search continues for the optimal cell type that can promote “true cardiac regeneration,” supplement the lost cardiomyocytes, and at the same time form the angiogenic support structure. One of the first stem cell types to be used for cardiac regeneration was derived from the mononuclear fraction of the bone marrow and the cells were designated as bone marrow mononuclear cells [1]. Ensuing years saw establishment of the finding that the bone marrow is host to a variety of distinct stem/progenitor cell populations possessing cardiogenic repair potential. The study by D. Orlic et al. in the early part of the last decade showed that transplantation of lin−/c-kit+ cells effectively transdifferentiates into cardiac lineages leading to enhanced cardiac function after infarction [2]. At the same time, a large body of evidence pointed towards a role for bone marrow derived mesenchymal stem cells (MSCs) in the repair of the damaged heart [3]. Nevertheless, the search for a heart resident stem cell population continued till A. P. Beltrami et al. showed that the adult heart contains a resident stem cell population capable of differentiating into all three cardiac lineages (myocytes, endothelial, and smooth muscle cells) [4]. In addition to these widely studied stem cell types, researchers have used other extra cardiac stem cell types such as adipose derived stem cells [5], cortical bone derived stem cells [6], and cord blood stem cells for cardiac repair. Despite the excitement, significant concerns persist around the ability of adoptively transferred cells to survive in the ischemic heart and some reports suggest as low as 1% of cells make it in the heart past the first few days of transplantation. Most of the salutary effects of cell therapy have been attributed to these few surviving cells and recent efforts have focused on boosting the survival, proliferation, and cardiac commitment of the donated stem cell population. This special issue contains a cluster of articles focused on augmenting ability of adoptively transferred stem cells to repair the damaged heart. X. Xue et al. demonstrated that genetic engineering of MSCs with Bcl-xl enhances survival and engraftment after transplantation leading to reduction in scar formation and increased functional recovery. Homing and migration of the adoptively transferred stem cells are another important determinant for the success of cell therapy. An interesting approach was employed using the ultrasound microbubble technique to promote MSC migratory ability to the infarcted myocardium. Authors show that ultrasound microbubble destruction increased MSC numbers in the infarcted heart mediated via SDF-1/CXCR4 axis. In contrast, the study by J. Liu et al. utilized adipose derived stem cells to show that curcumin, a naturally occurring food chemical, can promote angiogenic and survival ability of the cells augmenting their potential for the repair of ischemia reperfusion injury to the heart. Bone marrow derived endothelial progenitor cells (EPCs) represent a widely used cell type for cardiac regenerative therapies. EPCs have been shown to increase angiogenesis and augment cardiac function animal studies and undergoing clinical trials [7, 8]. EPCs remain one of the cells of choice for cardiac repair; however, the effect of different environmental factors remains to be fully elucidated. This special issue contains a very interesting finding about how low dose space-type ionizing radiation affects the long term cycling and survival of bone marrow derived EPCs opening up a host of possibilities in understanding how environmental factors influence stem cell function. Additionally, controversy rages on whether the transplanted stem cells directly convert into cardiomyocytes or mediate their beneficial effects through the release of “paracrine effectors.” Characterization of the stem cell secretome has provided some interesting clues about how factors released by the cells modulate cellular processes in target cells. These paracrine factors include growth factors, chemokine, cytokines, various proteins, and small molecules. Recently, small microvesicles released by stem cells under different physiological conditions have been included in the definition for paracrine factors. Exosomes are tiny vesicles released by the cells, carry proteins, mRNAs, and microRNA, and have the ability to modulate cellular and molecular signaling processes [9]. A number of recent studies have highlighted that exosomes derived from stem cells possess cardiac repair potential and are able to recapitulate the salutary effects of cell therapy promoting cardiac morphological and physiological functions [10, 11]. In this special issue, MSC derived exosomes and their role in cardiac regeneration have been highlighted by a report and a detailed review on MSC secretome. The study by K. Kang et al. demonstrates the therapeutic value of exosomes derived from CXCR4 overexpressing MSCs for the repair of heart after myocardial infarction. In addition, the review by C. Gallina et al. on MSC secretome and its potential application for cardiac regeneration is a wonderful collection of past and current research on MSC secreted factors and extracellular vesicles We hope the readers will find this special issue interesting and a timely effort covering current issues and advancements in the field of stem cell based therapies for the repair of damaged heart. Sadia Mohsin Daniele Avitabile Mohsin Khan