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Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes., Heart failure with preserved ejection fraction (HFpEF) is a global, major health issue for which no effective therapies are available. Here, the authors discover that the interplay between two transcription factors, Xbp1s and FoxO1, is critical for metabolic adaptation and lipid handling in HFpEF-stressed cardiomyocytes.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure (HF). Limited insight into underlying mechanisms has culminated in the longstanding absence of evidence-based therapies capable of mitigating the substantial morbidity and mortality associated with the syndrome. Existing clinical and epidemiological evidence suggests that excessive body fat and lipid mishandling contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are unknown. We recently developed a novel, clinically relevant, murine model of HFpEF, uncovering suppression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) signaling pathway as a critical driver of HFpEF pathogenesis. Objectives: To define and manipulate mechanisms downstream of Xbp1s in HFpEF and decipher its cardioprotective actions. Methods and Results: In the myocardium of experimental HFpEF, we detected cardiomyocyte steatosis coupled with increases in the abundance and activity of FoxO1 (Forkhead box protein O1), a conserved transcription factor involved in cell metabolism. FoxO1 depletion, as well as Xbp1s over-expression, in cardiomyocytes each ameliorated the HFpEF phenotype and reduced myocardial lipid accumulation. Strikingly, forced expression of Xbp1s in cardiomyocytes triggered proteasomal degradation of FoxO1. Furthermore, we discovered that FoxO1 is ubiquitinated upon Xbp1s over-expression, and Xbp1s-induced proteasomal degradation of FoxO1 occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1), a protein we identified as a novel and direct transcriptional target of Xbp1s. Conclusions: Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Background: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. Methods: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. Results: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. Conclusions: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.

Mutations in the gene encoding polycystin-1 (PC1) underlie autosomal dominant polycystic kidney disease (ADPKD). ADPKD patients present with multiple cardiovascular co-morbidities believed to be caused by renal dysfunction. LV hypertrophy and diastolic dysfunction can manifest during childhood or in young adults prior to a formal diagnosis of hypertension, and evidence suggests that LV function is impaired in ADPKD patients with normal or moderately reduced kidney function. These facts suggest that cardiomyocyte-autonomous effects may contribute to the cardiovascular abnormalities seen in ADPKD. Contractile function (systolic and diastolic) measured by echo was significantly reduced in PC1 cKO ( Pkd1 F/F ;αMHC-Cre) mice compared with controls ( Pkd1 F/F ). PC1 cKO cardiomyocytes manifest impaired contractility and smaller and slower Ca 2+ transients. Using a multidimensional approach, we discovered that cardiomyocytes lacking PC1 have shorter action potentials (APD50/90) and decreased SERCA activity. These alterations impair EC-coupling and decrease SR Ca 2+ loading during pacing. Remarkably, square pulses under voltage clamp (-80 to +10 mV) produced Ca 2+ transients with similar amplitude between genotypes, which highlights that alterations in action potential (AP) duration drive most of the EC-coupling changes. PC1-deficient cardiomyocytes manifested an increase in outward K + currents (I to , I Kslow1/2 and I ss ) but not in inward currents (I K1 ). PC1 over-expression in HEK293T cells reduced the currents of heterologously expressed Kv4.3/2.1/1.5 channels. The inhibitory effects of PC1 on Kv4.3 currents were mediated by PC1-CT (C-terminus) through its coiled-coil domain (CCD). Interestingly, a naturally occurring human mutant PC1 R4228X , located in the CCD, manifested no suppressive effects on Kv4.3 channel. Finally, to begin to test for relevance to human pathology, we found that PC1 ablation reduces AP duration, and PC1-CT over-expression had the opposite effect in human stem cell-derived cardiomyocytes. Our findings uncover a novel role for PC1 controlling action potential duration and SERCA. PC1-deficient cardiomyocytes manifest impaired contractility, likely contributing to contractile dysfunction in ADPKD patients.

BACKGROUND: Polycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes. METHODS: Wild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations. RESULTS: PC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca2+ transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca2+ stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity. An increase in outward K+ currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1R4228X manifested no suppressive effects on Kv4.3 channel activity. CONCLUSIONS: Our findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility.

Background: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. Methods: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. Results: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor β-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. Conclusions: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor β-1–SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

To investigate the role of age of human pediatric cardiac progenitor cells (hCPCs) on ventricular remodeling, the authors injected neonate, infant, or child hCPCs into rats with right ventricular heart failure. Mechanisms including migration and proliferation assays, as suggested by computational modeling, showed improved chemotactic and proliferative capacity of neonatal hCPCs compared with infant or child hCPCs. Thus, the reparative potential of hCPCs is age-dependent., Children with congenital heart diseases have increased morbidity and mortality, despite various surgical treatments, therefore warranting better treatment strategies. Here we investigate the role of age of human pediatric cardiac progenitor cells (hCPCs) on ventricular remodeling in a model of juvenile heart failure. hCPCs isolated from children undergoing reconstructive surgeries were divided into 3 groups based on age: neonate (1 day to 1 month), infant (1 month to 1 year), and child (1 to 5 years). Adolescent athymic rats were subjected to sham or pulmonary artery banding surgery to generate a model of right ventricular (RV) heart failure. Two weeks after surgery, hCPCs were injected in RV musculature noninvasively. Analysis of cardiac function 4 weeks post-transplantation demonstrated significantly increased tricuspid annular plane systolic excursion and RV ejection fraction and significantly decreased wall thickness and fibrosis in rats transplanted with neonatal hCPCs compared with saline-injected rats. Computational modeling and systems biology analysis were performed on arrays and gave insights into potential mechanisms at the microRNA and gene level. Mechanisms including migration and proliferation assays, as suggested by computational modeling, showed improved chemotactic and proliferative capacity of neonatal hCPCs compared with infant/child hCPCs. In vivo immunostaining further suggested increased recruitment of stem cell antigen 1-positive cells in the right ventricle. This is the first study to assess the role of hCPC age in juvenile RV heart failure. Interestingly, the reparative potential of hCPCs is age-dependent, with neonatal hCPCs exerting the maximum beneficial effect compared with infant and child hCPCs. Significance Stem cell therapy for children with congenital heart defects is moving forward, with several completed and ongoing clinical trials. Although there are studies showing how children differ from adults, few focus on the differences among children. This study using human cardiac progenitor cells shows age-related changes in the reparative ability of cells in a model of pediatric heart failure and uses computational and systems biology to elucidate potential mechanisms.

Cardiovascular disease remains an extremely deadly and costly burden across the globe. Since the myocardium has a limited regenerative capacity, current therapies are restricted to pharmaceuticals, mechanical and electrical support devices, and synthetic materials. Decellularized extracellular matrices have strong potential to provide support to failing myocardium. Their native composition offers complex biochemical cues beyond what can be synthesized in the laboratory. Their physical versatility affords multiple application routes as entire recellularized organs, acellular or recellularized patches, or injectable materials that conform to the dimensions of the injury. Manipulating the materials allows for engineering their mechanical properties, such as stiffness and degradation kinetics, through crosslinking or building hybrid scaffolds. Decellularized matrices can also serve as delivery vehicles by harnessing native conjugation sites for bioactive molecules. When delivered with cells, the decellularized matrix improves cell retention, protects these cells from the harsh microenvironment of the injured tissue, and directs their behavior. Importantly, the materials mimic the native extracellular matrix and can be remodeled, adapting and growing with the repaired tissue.

Cardiovascular disease, including myocardial infarction, is the number one cause of death. Current treatments are palliative and slow the progression toward heart failure, but to not regenerate healthy tissue. Self-assembling peptides are biomimietic, readily produced, non-immunogenic and non-cytotoxic. They do not assemble into hydrogels until triggered, allowing them to be injected into the myocardium and providing opportunities for minimally invasive therapies. The ability to tune the mechanical and bioactive properties of self-assembling peptides will continue to make them readily adaptable for mimicking natural microenvironments. To date, a variety of growth factors and signaling moieties have been incorporated into self-assembling peptide hydrogels, enhancing cell behavior and tissue function. Furthermore, the hydrogels serve as delivery vehicles for cells in vivo and platforms for improved cell culture. In addition to a brief review of self-assembling peptides, we will discuss a variety of their approaches for myocardial infarction therapy. Moreover, we will assess approaches taken in other tissue and discuss how these could benefit therapies for myocardial infarction.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Rationale: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects. Objective: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions. Methods and Results: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-β–stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue–signal–response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia–reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis. Conclusions: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using N

Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2–4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.

Cell therapy techniques are a promising option for tissue regeneration; especially in cases such as heart failure where transplantation is limited by donor availability. Multiple cell types have been examined for myocardial regeneration, including mesenchymal stem cells (and other bone marrow-derived cells), induced pluripotent stem cells, embryonic stem cells, cardiosphere-derived cells, and cardiac progenitor cells (CPCs). CPCs are multipotent and clonogenic, can be harvested from mature tissue, and have the distinct advantages of autologous transplant and lack of tumor formation in a clinical setting. Here we focus on the isolation, expansion, and myocardial differentiation of rat CPCs. Brief adaptations of the protocol for isolation from mouse and human tissue are also provided.

Cell therapy techniques are a promising option for tissue regeneration; especially in cases such as heart failure where transplantation is limited by donor availability. Multiple cell types have been examined for myocardial regeneration, including mesenchymal stem cells (and other bone marrow-derived cells), induced pluripotent stem cells, embryonic stem cells, cardiosphere-derived cells, and cardiac progenitor cells (CPCs). CPCs are multipotent and clonogenic, can be harvested from mature tissue, and have the distinct advantages of autologous transplant and lack of tumor formation in a clinical setting. Here we focus on the isolation, expansion, and myocardial differentiation of rat CPCs. Brief adaptations of the protocol for isolation from mouse and human tissue are also provided.

Introduction: Cell therapies have grown in popularity for myocardial regeneration post-infarction, but still suffer from poor retention, maturation and integration of delivered cells. Mechanical strain has been shown to alter cell size, shape, adherence and gene expression in cardiac cells. As a more recently identified cell type, the effect of mechanical strain on cardiac progenitor cells (CPCs) is unknown. This work aims to elucidate the role mechanical strain plays in CPC phenotype and if this response is matrix protein specific. We hypothesize that mechanical strain will improve CPC alignment and potential for connectivity. Methods: To examine the role of mechanical strain on CPCs, CPCs were seeded on FlexCell plates in the presence of a naturally-derived cardiac extracellularmatrix (cECM), collagen I (COL) or no protein (TCP) and strained 0% (static) or 10% at 1 Hz for 24 hours in a BioFlex system. CPC elongation, alignment, and size were evaluated by rhodamine-phalloidin staining. Connexin-43 expression was measured by Western and normalized to GAPDH. Data were analyzed by two-way ANOVA and Bonferroni post-test. Results: CPC area, independent of culture conditions, was 1020 ± 40 um2, corresponding to neonatal cardiomyocyte area. The aspect ratio (major/minor axis) of CPCs showed a trend for increased elongation with strain at (e.x. 2.0±0.2 for unstrained cECM compared to 2.7±0.1 for strained cECM; n=4, p>0.05). Static culture conditions, independent of matrix coating, showed 20±3% alignment of CPCs. Under strain, alignment increased to 30±2% on COL (n=4; p>0.05 for strained COL verus static COL) and 48±8% on cECM (n=4; p< 0.01 for strained cECM versus strained COL and p2 for connexin-43 protein in strained versus static conditions, independent of matrix, was observed (n=2, p>0.05) and confirmed by immunocytochemistry. Conclusion: This work suggests that mechanical strain alters CPC phenotype. Increased strain-induced alignment appears to be matrix dependent. In conclusion, these studies provide insight into the role of both mechanical forces and biochemical responses in the function of CPCs; which could lead to improved outcomes following cellular transplantation.

Cardiovascular disease is the leading cause of death in the United States. Following acute myocardial infarction, there is dramatic myocyte loss, and the cells are replaced with a collagen scar. Cell therapy directed toward regenerating infarcted myocardium suffers from poor retention of cells, possibly because the conditions are not ideal to support injected cells. Many studies show the local microenvironment is a potent regulator of cell fate. Therefore, we hypothesize that culturing cardiac progenitor cells (CPCs) on a naturally derived, cardiac extracellular matrix (cECM) will increase their function. CPCs were isolated from rat cardiac tissue-homogenate with magnetic beads conjugated with c-kit antibodies. Porcine, cardiac extracellular matrix (cECM) was decellularized, digested, and lyophillized to be used as a coating material. CPCs were cultured on cECM and examined for cardiogenic lineage markers, survival, proliferation and adherence. Our results show that CPCs cultured on cECM proliferate better (2.9-fold) as compared to COL (2.3-fold; p

Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds have attempted to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit(+) cells and were grown on plates coated with either cECM or collagen I (Col). Our results show an increase in early cardiomyocyte markers on cECM compared with Col, as well as corresponding protein expression at a later time. CPCs show stronger serum-induced proliferation on cECM compared with Col, as well as increased resistance to apoptosis following serum starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with Col. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as providing potential mechanisms to overcome the shortcomings of naked cell therapy.

Cardiovascular disease, including myocardial infarction, is a leading cause of death worldwide. Though several pharmacological treatments for severe dysfunction exist, much recent work has focused on the transplantation of adult-derived stem and progenitor cells. Early acute functional improvements have been noted, however long-term clinical efficacy is hampered by poor cell survival and engraftment. While the current treatment is infusion into the coronary artery, biomaterials may play an important role in modulating implanted cell function. This work aims to establish the role that a naturally derived extracellular matrix plays in the differentiation of cardiac progenitor cells (CPCs) and their potentially protective enzymatic systems. To test this hypothesis, we cultured rat CPCs in a naturally derived porcine ECM (pECM) and compared it to Collagen I. Quantitative real-time PCR was used to assess expression of cardiac, endothelial and smooth muscle markers. Additionally, angiotensin receptor (AT1R and AT2R) and antioxidant gene expressions were evaluated to determine the protective qualities of pECM. Preliminary data at 2 days following LIF removal demonstrate an increase in the expression of cardiac lineage markers (Nkx-2.5, Gata-4, α-MHC, and troponin I) in pECM compared to collagen. Smooth muscle markers, smooth muscle α-actin and sm22α as well as the endothelial marker Flk1 were also increased in pECM samples. Increased expression was also seen for antioxidant genes GPX1, SOD1, SOD2 and catalase in pECM cultured cells. Culturing in pECM for 7 days demonstrated an increase in Flt-1 and α-myosin heavy chain, indicating a potential increase in cardiogenesis. Moreover a 60% reduction in AT1R gene expression was observed with no significant change in AT2R expression. Our data demonstrate that culturing CPCs in naturally derived matrices may provide protection and enhance differentiation compared to collagen (present in high amounts in scarred myocardium). Future work will further elucidate this protective effect of AT1R downregulation and antioxidant increases in functional studies. In conclusion, pECM may be a potential cell delivery scaffold in post-MI treatment given its protective nature and improved differentiation influence.