Hina W. Chaudhry

Cyclin A2 Mediates Cardiomyocyte Mitosis in the Postmitotic Myocardium

Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes. Therefore, the concept of stimulating myocyte mitotic divisions has dramatic implications for cardiomyocyte regeneration and hence, cardiovascular disease. Previous reports describing manipulation of cell cycle proteins have not shown induction of cardiomyocyte mitosis after birth. We now report that cyclin A2, normally silenced in the postnatal heart, induces cardiac enlargement because of cardiomyocyte hyperplasia when constitutively expressed from embryonic day 8 into adulthood. Cardiomyocyte hyperplasia during adulthood was coupled with an increase in cardiomyoctye mitosis, noted in transgenic hearts at all time points examined, particularly during postnatal development. Several stages of mitosis were observed within cardiomyocytes and correlated with the nuclear localization of cyclin A2. Magnetic resonance analysis confirmed cardiac enlargement. These results reveal a previously unrecognized critical role for cyclin A2 in mediating cardiomyocyte mitosis, a role that may significantly impact upon clinical treatment of damaged myocardium.

Therapeutic Delivery of Cyclin A2 Induces Myocardial Regeneration and Enhances Cardiac Function in Ischemic Heart Failure

Background— Heart failure is a global health concern. As a novel therapeutic strategy, the induction of endogenous myocardial regeneration was investigated by initiating cardiomyocyte mitosis by expressing the cell cycle regulator cyclin A2. Methods and Results— Lewis rats underwent left anterior descending coronary artery ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing cyclin A2 (n =32) or empty adeno-null (n =32). Cyclin A2 expression was characterized by Western Blot and immunohistochemistry. Six weeks after surgery, in vivo myocardial function was analyzed using an ascending aortic flow probe and pressure-volume catheter. DNA synthesis was analyzed by proliferating cell nuclear antigen (PCNA), Ki-67, and BrdU. Mitosis was analyzed by phosphohistone-H3 expression. Myofilament density and ventricular geometry were assessed. Cyclin A2 levels peaked at 2 weeks and tapered off by 4 weeks. Borderzone cardiomyocyte cell cycle activation was demonstrated by increased PCNA (40.1±2.6 versus 9.3±1.1; P<0.0001), Ki-67 (46.3±7.2 versus 20.4±6.0; P<0.0001), BrdU (44.2±13.7 versus 5.2±5.2; P<0.05), and phosphohistone-H3 (12.7±1.4 versus 0±0; P<0.0001) positive cells/hpf. Cyclin A2 hearts demonstrated increased borderzone myofilament density (39.8±1.1 versus 31.8±1.0 cells/hpf; P=0.0011). Borderzone wall thickness was greater in cyclin A2 hearts (1.7±0.4 versus 1.4±0.04 mm; P<0.0001). Cyclin A2 animals manifested improved hemodynamics: Pmax (70.6±8.9 versus 60.4±11.8 mm Hg; P=0.017), max dP/dt (3000±588 versus 2500±643 mm Hg/sec; P<0.05), preload adjusted maximal power (5.75±4.40 versus 2.75±0.98 mWatts/&mgr;L2; P<0.05), and cardiac output (26.8±3.7 versus 22.7±2.6 mL/min; P=0.004). Conclusions— A therapeutic strategy of cyclin A2 expression via gene transfer induced cardiomyocyte cell cycle activation yielded increased borderzone myofilament density and improved myocardial function. This approach of inducing endogenous myocardial regeneration provides proof-of-concept evidence that cyclin A2 may ultimately serve as an efficient, alternative therapy for heart failure.

Cyclin A2 Induces Cardiac Regeneration After Myocardial Infarction and Prevents Heart Failure

Mammalian myocardial infarction is typically followed by scar formation with eventual ventricular dilation and heart failure. Here we present a novel model system in which mice constitutively expressing cyclin A2 in the myocardium elicit a regenerative response after infarction and exhibit significantly limited ventricular dilation with sustained and remarkably enhanced cardiac function. New cardiomyocyte formation was noted in the infarcted zones as well as cell cycle reentry of periinfarct myocardium with an increase in DNA synthesis and mitotic indices. The enhanced cardiac function was serially assessed over time by MRI. Furthermore, the constitutive expression of cyclin A2 appears to augment endogenous regenerative mechanisms via induction of side population cells with enhanced proliferative capacity. The ability of cultured transgenic cardiomyocytes to undergo cytokinesis provides mechanistic support for the regenerative capacity of cyclin A2.

Fetal Cells Traffic to Injured Maternal Myocardium and Undergo Cardiac Differentiation

Rationale: Fetal cells enter the maternal circulation during pregnancy and may persist in maternal tissue for decades as microchimeras. Objective: Based on clinical observations of peripartum cardiomyopathy patients and the high rate of recovery they experience from heart failure, our objective was to determine whether fetal cells can migrate to the maternal heart and differentiate to cardiac cells. Methods and Results: We report that fetal cells selectively home to injured maternal hearts and undergo differentiation into diverse cardiac lineages. Using enhanced green fluorescent protein (eGFP)-tagged fetuses, we demonstrate engraftment of multipotent fetal cells in injury zones of maternal hearts. In vivo, eGFP+ fetal cells form endothelial cells, smooth muscle cells, and cardiomyocytes. In vitro, fetal cells isolated from maternal hearts recapitulate these differentiation pathways, additionally forming vascular tubes and beating cardiomyocytes in a fusion-independent manner; ≈40% of fetal cells in the maternal heart express Caudal-related homeobox2 (Cdx2), previously associated with trophoblast stem cells, thought to solely form placenta. Conclusions: Fetal maternal stem cell transfer appears to be a critical mechanism in the maternal response to cardiac injury. Furthermore, we have identified Cdx2 cells as a novel cell type for potential use in cardiovascular regenerative therapy.

Cyclin A2 Induces Cardiac Regeneration After Myocardial Infarction Through Cytokinesis of Adult Cardiomyocytes

Cyclin A2 mediates cardiac regeneration of infarcted porcine hearts. A Change of Heart After Myocardial Infarction When blood flow is blocked off to the heart, the heart suffers permanent damage in part because cardiomyocytes are terminally differentiated and cannot proliferate. But what if these cells could be stimulated to divide? Some animals—like newts—have the ability to regenerate body parts when they are injured. Others—like zebrafish—can even regenerate heart tissue. Now, Shapiro et al. report that gene therapy can elicit a regenerative response in pig hearts. Cyclin A2 (Ccna2) has been shown to induce cardiac repair in small-animal models after myocardial infarction (MI). The authors have extended these studies by looking in the more translationally relevant pig model of MI. They found that Ccna2 delivered by an adenovirus improved heart function when compared with an adenoviral control. Cardiomyocytes in the pigs showed evidence of increased proliferation. If these data hold true in human studies, patients with MI can take heart. Cyclin A2 (Ccna2), normally silenced after birth in the mammalian heart, can induce cardiac repair in small-animal models of myocardial infarction. We report that delivery of the Ccna2 gene to infarcted porcine hearts invokes a regenerative response. We used a catheter-based approach to occlude the left anterior descending artery in swine, which resulted in substantial myocardial infarction. A week later, we performed left lateral thoracotomy and injected adenovirus carrying complementary DNA encoding CCNA2 or null adenovirus into peri-infarct myocardium. Six weeks after treatment, we assessed cardiac contractile function using multimodality imaging including magnetic resonance imaging, which demonstrated ~18% increase in ejection fraction of Ccna2-treated pigs and ~4% decrease in control pigs. Histologic studies demonstrate in vivo evidence of increased cardiomyocyte mitoses, increased cardiomyocyte number, and decreased fibrosis in the experimental pigs. Using time-lapse microscopic imaging of cultured adult porcine cardiomyocytes, we also show that Ccna2 elicits cytokinesis of adult porcine cardiomyocytes with preservation of sarcomeric structure. These data provide a compelling framework for the design and development of cardiac regenerative therapies based on cardiomyocyte cell cycle regulation.

Molecular physiology of cardiac regeneration

Heart disease is the leading cause of death in the industrialized world. This is partially attributed to the inability of cardiomyocytes to divide in a significant manner, and therefore the heart responds to injury through scar formation. One of the challenges of modern medicine is to develop novel therapeutic strategies to facilitate regeneration of cardiac muscle in the diseased heart. Numerous methods have been studied and a wide variety of cell types have been considered. To date, bone marrow stem cells, endogenous populations of cardiac stem cells, embryonic stem cells, and induced pluripotent stem cells have been investigated for their ability to regenerate infarcted myocardium, although stem cell transplantation has produced ambiguous results in human clinical trials. Several studies support another approach that seems very appealing: enhancing the limited endogenous regenerative capacity of the heart. The recent advances in stem cell and regenerative biology are giving rise to the view that cardiac regeneration, although not quite ready for clinical treatment, may translate into therapeutic reality in the not too distant future.

Engineered Cardiac Tissues for in vitro Assessment of Contractile Function and Repair Mechanisms

For efficiently assessing the potential for grafted cells to repair infarcted myocardium, a simplified surrogate heart muscle system would offer numerous advantages. Using neonatal rat cardiac myocytes in a collagen matrix, we created thin cylindrical engineered cardiac tissues (ECTs) that exhibit essential aspects of physiologic cardiac muscle function. Furthermore, a novel cryo-injured ECT model of myocardial infarction offers the potential for the longitudinal study of mechanisms of cell-based cardiac repair in vitro

Thymosin β4 and Cardiac Regeneration: Are We Missing a Beat?

Epicardial resident stem cells are known to differentiate into cardiomyocytes during cardiac development, amongst other cell types. Whether epicardium-derived progenitor cells (EPDCs) retain this plasticity in the adult heart has been the topic of heated scientific debate. Priming with thymosin beta 4, a peptide which has been suggested to be critical for cardiac development and to have cardio-protective properties, was recently shown to induce differentiation of EPDCs into cardiomyocytes in a small animal model of myocardial infarction. This finding is in stark contrast to another recent study in which thymosin beta 4 treatment following myocardial infarction did not induce cardiomyocyte differentiation of EPDCs. While EPDCs seem to exhibit overall cardio-protective effects on the heart following myocardial infarction, they have not been shown to differentiate into cardiomyocytes in a clinically relevant setting. It will be important to understand why the ability of one therapeutic agent to induce cardiomyocyte differentiation of EPDCs seemingly depends on a single variable, i.e. the time of administration. Furthermore, in light of a recent report, it appears that thymosin beta 4 may be dispensable for cardiac development.

Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes

This unit describes a streamlined two‐step protocol for the isolation of adult murine cardiomyocytes with subsequent Chromatin ImmunoPrecipitation (ChIP). Isolation and culturing of cardiomyocytes is a delicate process and the protocol presented here optimizes the combination of cardiomyocyte isolation with ChIP. ChIP is an invaluable method for analyzing molecular interactions occurring between a specific protein (or its post‐translationally modified form) and a region of genomic DNA. ChIP has become a widely used technique in the last decade since several groundbreaking studies have focused attention on epigenetics and have identified many epigenetic regulatory mechanisms. However, epigenetics within cardiovascular biology is a new area of focus for many investigators, and we have optimized a method for performing ChIP in adult murine cardiomyocytes, as we feel this will be an important aid to both the cardiovascular field and for the development of cell‐ and tissue‐specific ChIP. Curr. Protoc. Cell Biol. 58:17.14.1‐17.14.16.

Fetal Cells Traffic to Injured Maternal Myocardium and Undergo Cardiac DifferentiationNovelty and Significance

Rationale: Fetal cells enter the maternal circulation during pregnancy and may persist in maternal tissue for decades as microchimeras. Objective: Based on clinical observations of peripartum cardiomyopathy patients and the high rate of recovery they experience from heart failure, our objective was to determine whether fetal cells can migrate to the maternal heart and differentiate to cardiac cells. Methods and Results: We report that fetal cells selectively home to injured maternal hearts and undergo differentiation into diverse cardiac lineages. Using enhanced green fluorescent protein (eGFP)-tagged fetuses, we demonstrate engraftment of multipotent fetal cells in injury zones of maternal hearts. In vivo, eGFP+ fetal cells form endothelial cells, smooth muscle cells, and cardiomyocytes. In vitro, fetal cells isolated from maternal hearts recapitulate these differentiation pathways, additionally forming vascular tubes and beating cardiomyocytes in a fusion-independent manner; ≈40% of fetal cells in the maternal heart express Caudal-related homeobox2 (Cdx2), previously associated with trophoblast stem cells, thought to solely form placenta. Conclusions: Fetal maternal stem cell transfer appears to be a critical mechanism in the maternal response to cardiac injury. Furthermore, we have identified Cdx2 cells as a novel cell type for potential use in cardiovascular regenerative therapy.