Cardiac Myocyte Proliferation Research Articles

Gene expression and cellular changes in injured myocardium of Ciona intestinalis.

Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages for the study of cardiovascular biology. The regulatory mechanisms of cardiac myocyte proliferation in Ciona are intriguing since regeneration of functional tissue has been demonstrated in other organs of Ciona in response to injury. To identify genes that are differentially expressed in response to Ciona cardiac injury, microarray analysis was conducted on RNA from adult Ciona hearts with normal or damaged myocardium. After a 24- or 48-h recovery period, total RNA was isolated from damaged and control hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in comparison to control hearts. Ligation injury shows differential expression of 223 genes as compared to control with limited false discovery (5.8%). Among these 223 genes, 117 have known human orthologs of which 68 were upregulated and 49 were downregulated. Notably, Fgf9/16/20, insulin-like growth factor binding protein and Ras-related protein Rab11b were significantly upregulated in injured hearts, whereas expression of a junctophilin ortholog was decreased. Histological analyses of injured myocardium were conducted in parallel to the microarray study which revealed thickened myocardium in injured hearts. Taken together, these studies will connect differences in gene expression to cellular changes in the myocardium of Ciona, which will help to promote further investigations into the regulatory mechanisms of cardiac myocyte proliferation across chordates.

Fueling cardiac myocyte proliferation.

Fueling cardiac myocyte proliferation.

Development of Biodegradable Small Diameter Vascular Graft

Coronary heart disease (CHD) is a major cause of death on a global scale. Treatment options include revascularisation, but achieving a successful vascular graft requires using one with a diameter of less than 4mm and there is no artificial vascular graft at such a small diameter available. Scientists are interested in developing biodegradable artificial vascular grafts that can help overcome challenges associated with CHD. Associate Professor Kyohei Oyama and his team in the Department of Cardiac Surgery, Asahikawa Medical University, Japan, are currently working on two main projects: Creating a biodegradable small diameter vascular graft; and developing a heart regeneration treatment via endogenous cardiac myocyte proliferation. In developing a biodegradable small diameter vascular graft, the researchers hope to benefit patients that require the revascularisation of small vessels and also positively impact young patients who are still growing. The team also hope that the discoveries made through this research might lead to advances in other organ regeneration. When it comes to the need to recover lost cardiac tissue, this is unable to regenerate as cardiac myocytes (muscle cells) have no ability to proliferate. Oyama and the team are investigating the mechanism that limits this proliferation ability with a view to activating it. If cardiac myocyte proliferation can be activated during the revascularisation of CHD, then the damaged heart can be completely cured and the possibility of heart failure eliminated.

Myocardial injection of thermosensitive HADOPA/Ca-FVI/Alg hydrogels improves cardiac repair after myocardial infarction

Myocardial infarction (MI) is a common heart emergency that potentially causes death. Injectable hydrogels with excellent malleability and filling properties are suitable for myocardial injection and can avoid the need for open surgery. In this study, dopamine grafted hyaluronic acid (HADOPA) was synthesized and combined with sodium alginate (Alg) and Ca2+-loaded liposomes (Ca-FVI) to prepare a series of injectable thermosensitive sodium hyaluronate/sodium alginate hydrogels (HADOPA/Ca-FVI/Alg). At 37 °C, the HADOPA/Ca-FVI/Alg solution rapidly changed from liquid to hydrogel because of the crosslinking between sodium alginate and the Ca2+ released from Ca-FVI at a phase transition time of 100–200 s. The HADOPA/Ca-FVI/Alg scaffolds exhibited a porous 3D network structure, controlled porosity, and connected pore properties. Increasing the proportion of HADOPA increases the elasticity and absorbance of hydrogels. Experimental assessment showed that the hydrogels have good biocompatibility and that Alg1-HADOPA is more conducive to the adhesion, proliferation, and spreading of mouse cardiac myocytes while maintaining the best cell state. These findings suggest that HADOPA/Ca-FVI/Alg hydrogels improve cell proliferation and tissue repair, and that they are a promising therapeutic option for cardiac repair after MI.

Impact of Non-Pharmacological Interventions on the Mechanisms of Atherosclerosis.

Atherosclerosis remains the leading cause of mortality and morbidity worldwide characterized by the deposition of lipids and fibrous elements in the form of atheroma plaques in vascular areas which are hemodynamically overloaded. The global burden of atherosclerotic cardiovascular disease is steadily increasing and is considered the largest known non-infectious pandemic. The management of atherosclerotic cardiovascular disease is increasing the cost of health care worldwide, which is a concern for researchers and physicians and has caused them to strive to find effective long-term strategies to improve the efficiency of treatments by managing conventional risk factors. Primary prevention of atherosclerotic cardiovascular disease is the preferred method to reduce cardiovascular risk. Fasting, a Mediterranean diet, and caloric restriction can be considered useful clinical tools. The protective impact of physical exercise over the cardiovascular system has been studied in recent years with the intention of explaining the mechanisms involved; the increase in heat shock proteins, antioxidant enzymes and regulators of cardiac myocyte proliferation concentration seem to be the molecular and biochemical shifts that are involved. Developing new therapeutic strategies such as vagus nerve stimulation, either to prevent or slow the disease’s onset and progression, will surely have a profound effect on the lives of millions of people.

Abstract P2020: A Novel Molecular Mechanism Of Cardiac Hypertrophy

Proliferation of cardiac myocytes is accompanied by increases in cell mass to maintain myocyte size in fetal heart. In adult cardiac myocytes, cell growth becomes uncoupled from proliferation after growth stimuli, resulting in hypertrophic growth. Both proliferation and hypertrophy growth of cardiac myocytes require more protein synthesis. Brf1 (TF IIB-related factor 1) and Pol III genes (RNA Polymerase III-dependent genes) are the dominant factors of protein produce. Brf1 is a key regulator for Pol III genes. Cardiac hypertrophy is a critical risk factor for cardiovascular disease, including heart failure, arrhythmia, and sudden death. Cardiac hypertrophy involves both the processes of embryonic gene expression and transcriptional reprogramming. The processes are tightly modulated by different signal pathways and epigenetic regulation. Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. To explore the molecular mechanism of cardiac hypertrophy, we investigate the alterations of Brf1 and Pol III gene transcription under glucose treatment by using cell culture model and animal model. We have found that glucose increases Brf1 expression and Pol III gene transcription. We identified that these genes are mediated by JNK1, MSK1, pAMPK and phosphorylated histone H3. inhibiting the pathways decrease Brf1 expression, reduce Pol III gene transcription, consequently repression of myocytes growth. It implies that developing an effective inhibitor to repress Brf1 expression may provide a potential approach for the therapy of cardiac hypertrophy. More studies on cardiac hypertrophy are going in our laboratory.

RNA Sequencing of Cardiac in a Rat Model Uncovers Potential Target LncRNA of Diabetic Cardiomyopathy.

Background: Diabetic cardiomyopathy (DCM) is one of the major causes of heart failure in diabetic patients; however, its pathogenesis remains unclear. Long non-coding RNAs (lncRNAs) are involved in the development of various cardiovascular diseases, but little is known in DCM. Objective: The present study was conducted to investigate the altered expression signature of lncRNAs and mRNAs by RNA-sequencing and uncovers the potential targets of DCM. Methods: A DCM rat model was established, and the genome-wide expression profile of cardiac lncRNAs and mRNAs was investigated in the rat model with and without DCM by RNA-sequencing. Bioinformatics analysis included the co-expression, competitive endogenous RNA (ceRNA) network, and functional enrichment analysis of deregulated lncRNAs and mRNAs. Results: A total of 355 lncRNA transcripts and 828 mRNA transcripts were aberrantly expressed. The ceRNA network showed that lncRNA XR_351927.3, ENSRNOT00000089581, XR_597359.2, XR_591602.2, and XR_001842089.1 are associated with the greatest number of differentially expressed mRNAs and AURKB, MELK, and CDK1 may be the potential regulatory targets of these lncRNAs. Functional analysis showed that these five lncRNAs are closely associated with fibration, cell proliferation, and energy metabolism of cardiac myocytes, indicating that these core lncRNAs have high significance in DCM. Conclusions: The present study profiled the DCM-specific lncRNAs and mRNAs, constructed the lncRNA-related ceRNA regulatory network, and identified the potential prognostic biomarkers, which provided new insights into the pathogenesis of DCM.

The Hippo pathway regulates density-dependent proliferation of iPSC-derived cardiac myocytes

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell–cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition.

Intracellular Development of Resident Cardiac Stem Cells: An Overlooked Phenomenon in Myocardial Self-Renewal and Regeneration.

At present, the approaches aimed at increasing myocardial regeneration after infarction are not available. The key question is the identity of cells capable of producing functional cardiac myocytes (CMs), replenishing those lost during ischemia. With identification of resident cardiac stem cells (CSCs), it has been supposed that this cell population may be crucial for myocardial self-renewal and regeneration. In the last few years, the focus has been shifted towards another concept, implying that new CMs are produced by dedifferentiation and proliferation of mature CMs. The observation that CSCs can undergo development inside immature cardiac cells by formation of “cell-in-cell structures” (CICSs) has enabled us to conclude that encapsulated CICSs are implicated in mammalian cardiomyogenesis over the entire lifespan. Earlier we demonstrated that new CMs are produced through formation of CSC-derived transitory amplifying cells (TACs) either in the CM colonies or inside encapsulated CICSs. In this study, we described the phenomenon of CSC penetration into mature CMs, resulting in the formation of vacuole-like CICSs (or non-encapsulated CICSs) containing proliferating CSCs with subsequent differentiation of CSC progeny into TACs and their release. In addition, we compared the phenotypes of TACs derived from encapsulated and non-encapsulated CICSs developing in immature and mature CMs, respectively.

Cardioprotection Generated by Aerobic Exercise Training is Not Related to the Proliferation of Cardiomyocytes and Angiotensin-(1-7) Levels in the Hearts of Rats with Supravalvar Aortic Stenosis

The beneficial effect of aerobic exercise training (ET) on cardiac remodeling caused by supravalvar aortic stenosis (AS) has been demonstrated in experimental studies; however, the mechanisms responsible for improving cardiac function are not entirely understood. We evaluated whether ET-generated cardioprotection in pressure-overloaded rats is dependent on cardiomyocyte proliferation, increased angiotensin-(1-7) (Ang-1-7) levels, and its receptor in the myocardium. Eighteen weeks after ascending AS surgery, Wistar rats were randomly assigned to four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary aortic stenosis (AS-Sed) and exercised aortic stenosis (AS-Ex) groups. The moderate treadmill exercise protocol was performed for ten weeks. The functional capacity was assessed by treadmill exercise testing. Cardiac structure and function were evaluated by echocardiogram. Cardiomyocyte proliferation was evaluated by flow cytometry. Expression of cell cycle regulatory genes as CCND2, AURKB, CDK1, and MEIS1 was verified by RT-qPCR. Cardiac and plasma angiotensin I (Ang I), angiotensin II (Ang II), and Ang-(1-7) levels were analyzed by high-performance liquid chromatography (HPLC). The angiotensin-converting enzyme (ACE) activity was assessed by the fluorometric method and protein expression of AT1 and Mas receptors by Western blot. The AS-Ex group showed reduced left ventricular wall relative thickness and improved ejection fraction; also, it showed decreased gene expression of myocyte cell cycle regulators, ACE, Ang I, Ang II and Ang II/Ang-(1-7) ratio levels compared to AS-Sed group. However, ET did not induce alterations in Ang-(1-7) and cardiac Mas receptor expression and myocyte proliferation. Aerobic exercise training improves systolic function regardless of myocyte proliferation and Ang-(1-7)/Mas receptor levels. However, the ET negatively modulates the vasoconstrictor/hypertrophic axis (ACE/Ang II) and decreases the expression of negative regulatory genes of the cell cycle in cardiomyocytes of rats with supravalvular aortic stenosis.

Acute inflammation contributes to regeneration in the neonatal porcine heart

The adult mammalian heart has limited ability to repair itself following injury. Zebrafish, newts and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation.

Non-coding RNAs in Cardiac Regeneration.

Cardiovascular disease is a leading cause of death worldwide, and with the dramatically increasing numbers of heart failure patients in the next 10years, mortality will only increase [1]. For patients with end-stage heart failure, heart transplantation is the sole option. Regrettably, the number of available donor hearts is drastically lower than the number of patients waiting for heart transplantation. Despite evidence of cardiomyocyte renewal in adult human hearts, regeneration of functional myocardium after injury can be neglected. The limited regenerative capacity due to inadequate proliferation of existing cardiomyocytes is insufficient to repopulate areas of lost myocardium [2]. As a solution, the hypothesis that adult stem cells could be employed to generate functional cardiomyocytes was proposed. One of the early studies that supported this hypothesis involved direct injection of hematopoietic c-kit-positive cells derived from bone marrow into the infarcted heart [3]. However, in sharp contrast, more recent evidence emerged demonstrating that these hematopoietic stem cells only differentiate into cells down the hematopoietic lineage rather than into cardiomyocytes [4, 5], and the focus shifted towards stem cells residing in the heart, called cardiac progenitor cells. These CPCs were extracted and injected into the myocardium to regenerate the heart [6]. In recent years, over 80 pre-clinical studies employing cardiac stem cells in vivo in large and small animals to evaluate the effect on functional parameters were systematically reviewed, identifying differences between large and small animals [7]. Despite the positive outcome of these stem cell therapies on functional parameters, c-kit-positive cardiac progenitor cells were shown to contribute minimally to the generation of functional cardiomyocytes [8, 9]. This heavily debated topic is summarized concisely by van Berlo and Molkentin [10]. Recently, single-cell sequencing and genetic lineage tracing of proliferative cells in the murine heart in both homeostatic and regenerating conditions did not yield a quiescent cardiac stem cell population or other cell types that support transdifferentiation into cardiomyocytes, nor did it support proliferation of cardiac myocytes [11, 12]. Now, the focus is shifting towards exploiting the limited regenerative capacity of the cardiomyocytes themselves, by re-activating proliferation of existing cardiomyocytes through dedifferentiation, reentry into the cell cycle, and cytokinesis. This process is the new focus of research to promote cardiac regeneration, and can be controlled on multiple levels, including cell-cycle manipulation, reprogramming, small molecules, extra-cellular matrix (ECM), proteins, and RNA regulation [13].

Abstract 153: Dexamethasone Inhibits Regeneration and Causes Ventricular Aneurysm in the Neonatal Porcine Heart After Myocardial Infarction

Background: The adult mammalian heart has limited ability to repair itself following injury. Zebrafish, newts and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. More recently, we demonstrated that the hearts of neonatal pigs (2-day old) have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth. The current study aims to determine whether acute inflammation is required for CM regeneration after myocardial infarction (MI). Methods: We examined the role of acute inflammation on regenerative capacity of the neonatal pig heart (ages: 2 days postnatal) by intramuscular injection of dexamethasone (Dex) during the first week post-MI surgery. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance (CMR) imaging. Bromodeoxyuridine pulse-chase labeling, histology, and immunohistochemistry were performed to study CM cytokinesis and to quantify myocardial fibrosis. Results: After MI, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day old pigs. In contrast, piglets received Dex injection developed ventricular aneurysm and did not recover function. Dex limited CM cytokinesis via inhibiting aurora-B protein expression and caused mature scar and thinned walls at infarct site. Conclusions: Dex suppresses acute inflammation, inhibits CM regeneration, and causes ventricular aneurysm in neonatal pig hearts after MI.

Notch1 Overexpression in Cardiac Mesenchymal Stem Cells Renders their Exosomes Highly Effective in Promoting Angiogenesis and Cardiac Regeneration

ObjectiveNotch1 signaling is critical in postnatal angiogenesis. Cardiac mesenchymal stem cells (C‐MSC) are endogenous cardiac stromal cells which are believed to facilitate cardiac regeneration via paracrine effects. Here, we investigated the effects of exosomes from Notch1 engineered C‐MSC on angiogenesis and cardiac myocyte proliferation in ischemic myocardium.Methods and resultsC‐MSC were isolated from conditional Notch1 knock out mice. For Notch1 knockout C‐MSC generation, Notch1 gene was deleted via adenoviral vector mediated Cre recombination. For Notch1 overexpression, cells were modified with Notch1 intracellular domain (NICD). Myocardial infarction (MI) mouse model was generated by LAD ligation. Exosomes from C‐MSC (Exo‐C‐MSC), Notch1 knockout C‐MSC (Exo‐C‐MSCNICD KO) and Notch1 overexpressing C‐MSC (Exo‐C‐MSCNICD) were isolated and characterized. Both Exo‐C‐MSC and Exo‐C‐MSCNICD were strongly pro‐angiogenic under in vitro and in vivo conditions. Compared with Exo‐C‐MSC, Exo‐C‐MSCNICD treatment led to dense tube formation and higher vessel density in peri‐infarct area. Moreover, knockout of Notch1 significantly abrogated the pro‐angiogenetic effect of Exo‐C‐MSC. Compared with control group, pretreatment of Exo‐C‐MSC and Exo‐C‐MSCNICD also exerted anti‐apoptotic effects on endothelial cells stressed with 300 μM H2O2, for 8h. Exo‐C‐MSCNICD KO had no anti‐apoptotic effect in H202 treated cells. As expected, intramyocardial injection of Exo‐C‐MSCNICD were strongly cardioprotective on cardiomyocytes (CMs) and vessel cells 24h after MI. Though both Exo‐C‐MSC and Exo‐C‐MSCNICD treatment improved cardiac function and decreased fibrosis in mice one‐month post‐MI, Exo‐C‐MSCNICD were more effective in improving cardiac function and reducing fibrosis. On the other hand, Exo from Notch1 knockout MSC had no effects on fibrosis or function. A significant effect on vessel density and CMs proliferation (ki67 and α‐actinin double positive cells) in peri infarct area was observed with the treatment of Exo‐C‐MSCNICD in comparison with PBS, Exo‐C‐MSC and Exo‐C‐MSCNICD KO groups.ConclusionNotch1 overexpression in C‐MSC produced highly effective exosomes in preventing cell death, promoting angiogenesis, cardiomyocyte proliferation and in restoring cardiac function after MI. These data suggest that Notch1 activation may further enhance the effectiveness of exosomes from C‐MSC in cell‐free therapy.Support or Funding InformationR01 HL134354 and R01 AR070029This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Early Regenerative Capacity in the Porcine Heart.

The adult mammalian heart has limited ability to repair itself after injury. Zebrafish, newts, and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. It is unknown whether hearts of young large mammals can regenerate. We examined the regenerative capacity of the pig heart in neonatal animals (ages 2, 3, or 14 days postnatal) after myocardial infarction or sham procedure. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance imaging and echocardiography. Bromodeoxyuridine pulse-chase labeling, histology, immunohistochemistry, and Western blotting were performed to study cell proliferation, sarcomere dynamics, and cytokinesis and to quantify myocardial fibrosis. RNA-sequencing was also performed. After myocardial infarction, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day-old pigs. In contrast, older animals developed full-thickness myocardial scarring, thinned walls, and did not recover function. Genome-wide analyses of the infarct zone revealed a strong transcriptional signature of fibrosis in 14-day-old animals that was absent in 2-day-old pigs, which instead had enrichment for cytokinesis genes. In regenerating hearts of the younger animals, up to 10% of CMs in the border zone of the myocardial infarction showed evidence of DNA replication that was associated with markers of myocyte division and sarcomere disassembly. Hearts of large mammals have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth.

3D Carbon-Nanotube-Based Composites for Cardiac Tissue Engineering.

Heart failure is a disease of epidemic proportion and a leading cause of mortality in the world. Because cardiac myocytes are terminally differentiated cells with minimal intrinsic ability to self-regenerate, cardiac tissue engineering has emerged as one of the most realistic therapeutic strategies for cardiac repair. We have previously proven the ability of carbon nanotube scaffolds to promote cardiomyocyte proliferation, maturation, and long-term survival. Here, we tested if three-dimensional scaffolds of carbon nanotube-based composites can also promote cardiomyocyte growth, electrophysiological maturation, and formation of functional syncytia. To this purpose, we developed an elastomeric scaffold that consists of a microporous and self-standing material made of polydimethylsiloxane (PDMS) containing micrometric cavities, and integrated multiwall carbon nanotubes (MWCNTs) into the scaffold. We combined microscopy, cell biology, and calcium imaging to investigate whether neonatal rat ventricular myocytes (NRVMs) cultured on the 3D-PDMS+MWCNT acquire a more viable and mature phenotype compared to control. We found that when cultured in the 3D-PDMS+MWCNTs, NRVMs showed improved viability (p < 0.005 at day 3) and more defined and mature sarcomeric phenotype compared to 3D-PDMS control. These modifications were associated with an increase of connexin-43 gene expression, gap junction areas (p < 0.005 at day 3), and a more mature electrophysiological phenotype of syncytia and calcium transients. Finally, 3D-PDMS+MWCNT boosted NRVMs proliferation (p < 0.005 at day 3) while hindering cardiac fibroblasts proliferation compared to control PDMS. Thus, 3D-PDMS+MWCNT has the ability to promote viability, proliferation and functional maturation of cardiac myocytes. These properties are essential in cardiac tissue engineering and offer novel perspectives in the development of innovative therapies for cardiac repair.

Cardiac myocyte proliferation and maturation near term is inhibited by early gestation maternal testosterone exposure.

Polycystic ovary syndrome is a complex and common disorder in women, and those affected experience an increased burden of cardiovascular disease. It is an intergenerational syndrome, as affected women with high androgen levels during pregnancy "program" fetal development, leading to a similar phenotype in their female offspring. The effect of excess maternal testosterone exposure on fetal cardiomyocyte growth and maturation is unknown. Pregnant ewes received biweekly injections of vehicle (control) or 100 mg testosterone propionate between 30 and 59 days of gestation (early T) or between 60 and 90 days of gestation (late T). Fetuses were delivered at ~135 days of gestation, and their hearts were enzymatically dissociated to measure cardiomyocyte growth (dimensional measurements), maturation (proportion binucleate), and proliferation (nuclear Ki-67 protein). Early T depressed serum insulin-like growth factor 1 and caused intrauterine growth restriction (IUGR; P < 0.0005). Hearts were smaller with early T ( P < 0.001) due to reduced cardiac myocyte maturation ( P < 0.0005) and proliferation ( P = 0.017). Maturation was also lower in male than female fetuses ( P = 0.004) independent of treatment. Late T did not affect cardiac growth. Early excess maternal testosterone exposure depresses circulating insulin-like growth factor 1 near term and causes IUGR in both female and male offspring. These fetuses have small, immature hearts with reduced proliferation, which may reduce cardiac myocyte endowment and predispose to adverse cardiac growth in postnatal life. While excess maternal testosterone exposure leads to polycystic ovary syndrome and cardiovascular disease in female offspring, it may also predispose to complications of IUGR and cardiovascular disease in male offspring. NEW & NOTEWORTHY Using measurements of cardiac myocyte growth and maturation in an ovine model of polycystic ovary syndrome, this study demonstrates that early gestation excess maternal testosterone exposure reduces near-term cardiomyocyte proliferation and maturation in intrauterine growth-restricted female and male fetuses. The effect of testosterone is restricted to exposure during a specific period early in pregnancy, and the effects appear mediated through reduced insulin-like growth factor 1 signaling. Furthermore, male fetuses, regardless of treatment, had fewer mature cardiomyocytes than female fetuses.

Abstract 465: Short Term Hypoxia Can Induce Adult Feline Cardiomyocytes to Re-enter the Cell Cycle and Divide

Rationale: The mammalian adult heart has a very limited capacity to regenerate cardiomyocytes that die from pathological damage. Most recent studies suggest that those few new myocytes found after injury are derived from pre-existing cardiomyocytes. A recent study in mice suggest that systemic hypoxemia in adult animals can induce cardiac myocyte cell cycle reentry and myocyte proliferation. In the present experiments we evaluate the ability of adult feline cardiomyocytes to reenter the cell cycle and proliferate in hypoxic environments. Hypothesis: To determine if hypoxia induces adult feline cardiomyocytes to regenerate. Methods and results: Adult ventricular cardiomyocytes were isolated from adult felines and maintained in culture. For acute hypoxia, cultures were placed in a hypoxic chamber on day 1, 3, 5, 7, 10, or 14 following isolation and subjected to 1% O 2 , 5% CO 2 , and the balance was N 2 for 24 hours. For chronic hypoxia, adult cardiomyocytes were subjected to 1% O 2 , 5% CO 2 with the balance of N 2 at day 1 after isolation and kept at continuous hypoxia for 3, 5, 7, 10, or 14 days. Prior to hypoxia, cells were treated with 10uM EdU to identify new DNA in cardiomyocytes. Acute hypoxia was shown to promote cell division in cardiac myocytes at all time points except at day 1 post isolation when compared to control cultures. However, chronic hypoxia caused cardiomyocyte damage and increased cell death in all time points as compared to control and acute hypoxia cultures. Conclusion: Short term hypoxia can induce adult feline cardiomyocytes to proliferate, while long term exposure leads to injury and apoptosis.

Understanding Key Mechanisms of Exercise-Induced Cardiac Protection to Mitigate Disease: Current Knowledge and Emerging Concepts.

The benefits of exercise on the heart are well recognized, and clinical studies have demonstrated that exercise is an intervention that can improve cardiac function in heart failure patients. This has led to significant research into understanding the key mechanisms responsible for exercise-induced cardiac protection. Here, we summarize molecular mechanisms that regulate exercise-induced cardiac myocyte growth and proliferation. We discuss in detail the effects of exercise on other cardiac cells, organelles, and systems that have received less or little attention and require further investigation. This includes cardiac excitation and contraction, mitochondrial adaptations, cellular stress responses to promote survival (heat shock response, ubiquitin-proteasome system, autophagy-lysosomal system, endoplasmic reticulum unfolded protein response, DNA damage response), extracellular matrix, inflammatory response, and organ-to-organ crosstalk. We summarize therapeutic strategies targeting known regulators of exercise-induced protection and the challenges translating findings from bench to bedside. We conclude that technological advancements that allow for in-depth profiling of the genome, transcriptome, proteome and metabolome, combined with animal and human studies, provide new opportunities for comprehensively defining the signaling and regulatory aspects of cell/organelle functions that underpin the protective properties of exercise. This is likely to lead to the identification of novel biomarkers and therapeutic targets for heart disease.

Transplantation of cardiac stem cells overexpressing integrin-linked kinase improves cardiac function in a rat model of acute myocardial infarction

Objective: To investigate the effect of cardiac stem cells (CSC) overexpressing integrin-linked kinase (ILK-CSC) transplantation on cardiac function after myocardial infarction (MI) and related mechanism. Methods: CSCs were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and ILK-CSC were established by transfecting recombinant adenoviral vector harboring human wild-type ILK cDNA. Forty 8-week-old rats were randomly divided into 4 groups (n=10 each group): sham group, MI plus saline injection group(saline group), MI plus CSC transfected with null vector injection group (Ad-null-CSC group), and MI plus ILK-CSC injection group(ILK-CSC group). MI was induced through coronary artery ligation, and after 15 minutes, 30 μl saline, Ad-null-CSC (1×10(5) cells/30 μl) or ILK-CSC (1×10(5) cells/30 μl) were injected into the hearts of MI rats at 3 different points in infracted zone and infarct border zone. After 4 weeks, left ventricular (LV) function was examined by echocardiography, LV fibrosis was detected by HE and Masson staining, and myocardial protein expression of Ki-67 and p-H3 was evaluated by immuohistochemistry and mRNA expression of cyclinD1 and PCNA was detected by real-time RT-PCR. Results: (1) Thirty-seven rats (sham group=10, saline group=8, Ad-null-CSC group=9 and ILK-CSC group=10) survived at 4 weeks after operation. Left ventricular end-systolic dimension (LVESD, P=0.009) and left ventricular end-diastolic dimension (LVEDD, P=0.002) were significantly increased, and left ventricular ejection fraction(LVEF, P=0.006) was decreased in saline group compared with those of sham group.In Ad-null-CSC group LVESD (P=0.005) and LVEDD (P=0.003) were decreased, but LVEF remained unchanged (P=0.113) compared with those of saline group. LVESD (P=0.004) and LVEDD (P=0.000 1) of ILK-CSC group were significantly decreased, and LVEF (P=0.004) was significantly increased compared with those of Ad-null-CSC group. (2) LV histology and myocardial fibrosis: there were marked myocyte loss and significant increase of myocardial fibrosis in the saline group((70.6±5.1) %, P=0.002) and Ad-null-CSC group((57.7±3.4) %, P=0.001) compared with sham group ((8.2±2.2) %), while the fibrosis was significantly attenuated post injection of ILK-CSC ((30.6±7.0)%, P=0.005 vs. Ad-null-CSC). (3) Protein levels of mitotic genes: the results of immuohistochemistry showed that the brown positive particles were presented in the nuclei of cardiac myocytes in saline, Ad-null-CSC and ILK-CSC groups, but they were negative in sham group. The protein expression of Ki-67 (P=0.007) and phosphohistone-H3 (p-H3) (P=0.003) in ILK-CSC group was significantly higher than in Ad-null-CSC group. (4) mRNA levels of mitotic genes: the results of real-time RT-PCR showed that the mRNA levels of cyclinD1 and proliferating cell nuclear antigen (PCNA) in saline and Ad-null-CSC groups were similar as in sham group (all P>0.05). However, in ILK-CSC group they were significantly increased compared with those in Ad-null-CSC group (cyclinD1, P=0.003; PCNA, P=0.004). Conclusion: Our results suggest that myocardial transplantation of CSC overexpressing ILK improves cardiac function in the post-MI rats, and this beneficial effect may be related to the enhanced proliferation of cardiac myocytes and attenuated myocardial fibrosis.