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Fibroblast growth factor 2 (FGF2), produced as high (Hi-) and low (Lo-) molecular weight isoforms, is implicated in cardiac response to injury. The role of endogenous FGF2 isoforms during chronic stress is not well defined. We investigated the effects of endogenous Hi-FGF2 in a mouse model of simulated pressure-overload stress achieved by transverse aortic constriction (TAC) surgery. Hi-FGF2 knockout mice, expressing only Lo-FGF2, FGF2(Lo), and wild-type mice, FGF2(WT), expressing both Hi-FGF2 and Lo-FGF2, were used. By echocardiography, a decline in systolic function was observed in FGF2(WT) but not FGF2(Lo) mice compared to corresponding sham-operated animals at 4–8 weeks post-TAC surgery. TAC surgery increased markers of myocardial stress/damage including B-type natriuretic peptide (BNP) and the pro-cell death protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 (Bnip3) in FGF2(WT) but not FGF2(Lo) mice. In FGF2(Lo) mice, cardiac levels of activated FGF receptor 1 (FGFR1), and downstream signals, including phosphorylated mTOR and p70S6 kinase, were elevated post-TAC. Finally, NR1D1 (nuclear receptor subfamily 1 group D member 1), implicated in cardioprotection from pressure-overload stress, was downregulated or upregulated in the presence or absence, respectively, of Hi-FGF2 expression, post-TAC surgery. In wild-type cardiomyocyte cultures, endothelin-1 (added to simulate pressure-overload signals) caused NR1D1 downregulation and BNP upregulation, similar to the effect of TAC surgery on the FGF2(WT) mice. The NR1D1 agonist SR9009 prevented BNP upregulation, simulating post-TAC findings in FGF2(Lo) mice. We propose that elimination of Hi-FGF2 is cardioprotective during pressure-overload by increasing FGFR1-associated signaling and NR1D1 expression.

We have previously shown that overexpression of SKI, an endogenous TGF-β1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-β signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI’s effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI’s capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00865-9.

Heart disease with attendant cardiac fibrosis kills more patients in developed countries than any other disease, including cancer. We highlight the recent literature on factors that activate and also deactivate cardiac fibroblasts. Activation of cardiac fibroblasts results in myofibroblasts phenotype which incorporates aSMA to stress fibres, express ED-A fibronectin, elevated PDGFRα and are hypersecretory ECM components. These cells facilitate both acute wound healing (infarct site) and chronic cardiac fibrosis. Quiescent fibroblasts are associated with normal myocardial tissue and provide relatively slow turnover of the ECM. Deactivation of activated myofibroblasts is a much less studied phenomenon. In this context, SKI is a known negative regulator of TGFb1 /Smad signalling, and thus may share functional similarity to PPARγ activation. The discovery of SKI's potent anti-fibrotic role, and its ability to deactivate and/or myofibroblasts is featured and contrasted with PPARγ. While myofibroblasts are typically recruited from pools of potential precursor cells in a variety of organs, the importance of activation of resident cardiac fibroblasts has been recently emphasised. Myofibroblasts deposit ECM components at an elevated rate and contribute to both systolic and diastolic dysfunction with attendant cardiac fibrosis. A major knowledge gap exists as to specific proteins that may signal for fibroblast deactivation. As SKI may be a functionally pluripotent protein, we suggest that it serves as a scaffold to proteins other than R-Smads and associated Smad signal proteins, and thus its anti-fibrotic effects may extend beyond binding R-Smads. While cardiac fibrosis is causal to heart failure, the treatment of cardiac fibrosis is hampered by the lack of availability of effective pharmacological anti-fibrotic agents. The current review will provide an overview of work highlighting novel factors which cause fibroblast activation and deactivation to underscore putative therapeutic avenues for improving disease outcomes in cardiac patients with fibrosed hearts.

Two-dimensional cell culture is the primary method employed for proof-of-concept studies in most molecular biology labs. While immortalized cell lines are convenient and easy to maintain for extended periods in vitro, their inability to accurately represent genuine cell physiology-or pathophysiology-presents a challenge for drug discovery, as most results are not viable for the transition to clinical trial. The use of primary cells is a more biologically relevant approach to this issue; however, simulating in vitro what is observed in vivo is exigent at best. Primary cardiac fibroblasts are particularly difficult to maintain in a quiescent state, due to their innate phenotypic plasticity, and sensitivity to mechanical and biochemical stimulus. As conventional cell culture methods do not consider these factors, here we describe a method that limits environmental input (i.e., mechanical, nutritional, hormonal) to extend the physiological cardiac fibroblast phenotype in vitro.

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analogue misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

Cardiac fibroblast activation to hyper-synthetic myofibroblasts following a pathological stimulus or in response to a substrate with increased stiffness may be a key tipping point for the evolution of cardiac fibrosis. Cardiac fibrosis per se is associated with progressive loss of heart pump function and is a primary contributor to heart failure. While TGF-β is a common cytokine stimulus associated with fibroblast activation, a druggable target to quell this driver of fibrosis has remained an elusive therapeutic goal due to its ubiquitous use by different cell types and also in the signaling complexity associated with SMADs and other effector pathways. More recently, mechanical stimulus of fibroblastic cells has been revealed as a major point of activation; this includes cardiac fibroblasts. Further, the complexity of TGF-β signaling has been offset by the discovery of members of the SKI family of proteins and their inherent anti-fibrotic properties. In this respect, SKI is a protein that may bind a number of TGF-β associated proteins including SMADs, as well as signaling proteins from other pathways, including Hippo. As SKI is also known to directly deactivate cardiac myofibroblasts to fibroblasts, this mode of action is a putative candidate for further study into the amelioration of cardiac fibrosis. Herein we provide a synthesis of this topic and highlight novel candidate pathways to explore in the treatment of cardiac fibrosis.

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFβ occurs via a scleraxis-dependent mechanism: TGFβ induces recruitment of scleraxis to the MMP2 promoter, and TGFβ was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.

Background Normothermic ex vivo heart perfusion (EVHP) has been shown to improve the preservation of hearts donated after circulatory arrest and to facilitate clinical successful transplantation. Steroids are added to the perfusate solution in current clinical EVHP protocols; however, the impact of this approach on donor heart preservation has not been previously investigated. We sought to determine the impact of steroids on the inflammatory response and development of myocardial edema during EVHP. Methods Thirteen pigs were anesthetized, mechanical ventilation was discontinued, and a hypoxemic cardiac arrest ensued. A 15-minute warm-ischemic standoff period was observed, and then hearts were resuscitated with a cardioplegic solution. Donor hearts were then perfused ex vivo in a normothermic beating state for 6 hours with 500 mg of methylprednisolone (steroid: n = 5) or without (control: n = 8). Results The addition of steroids to the perfusate solution reduced the generation of proinflammatory cytokines (interleukin-6, -8, -1β, and tumor necrosis factor-α) and the development of myocardial edema during EVHP (percentage of weight gain: control = 26% ± 7% versus steroid = 16% ± 10%, p = 0.049). Electron microscopy suggested less endothelial cell edema in the steroid group (injury score: control = 1.8 ± 0.2 versus steroid = 1.2 ± 0.2, p = 0.06), whereas perfusate troponin-I (control = 11.9 ± 1.9 ng/mL versus steroid = 9.5 ± 2.4 ng/mL, p = 0.448) and myocardial function were comparable between the groups. Conclusions The addition of methylprednisolone to the perfusion solution minimizes the generation of proinflammatory cytokines and development of myocardial edema during normothermic ex vivo perfusion of hearts donated after circulatory arrest.

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-β1 (TGF-β1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-β1 induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-β1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-β1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

Fibroblast growth factor 2 (FGF2) is implicated in normal cardiac development as well as cardiac pathophysiology; however, FGF2 exist as multiple high and low molecular weight isoforms. While endogenous low molecular weight FGF2 (Lo-FGF2) is cardioprotective during chronic stress, the more prevalent endogenous high molecular weight FGF2 (Hi-FGF2) is proposed to promote maladaptive cardiac remodeling. We have investigated the hypothesis that genetic elimination of Hi-FGF attenuates cardiac dysfunction in mice that have been subjected to pressure overload by transverse aortic constriction (TAC). Two groups of male C57BL/6mice were compared: (1) Wild type (WT) mice, expressing Hi- and Lo-FGF2 (FGF[WT] mice); and (2) Hi-FGF2 knock-out mice, expressing only Lo-FGF2 (FGF[Lo] mice). Echocardiographic assessment of heart function and dimensions was done at baseline and then 4 and 8 weeks after TAC or sham surgery. FGF[WT] mice displayed a decline in systolic function compared to their corresponding sham animals at 4- and 8-weeks post-TAC, which was absent in the FGF[Lo] mice. Relative levels of B-type natriuretic peptide, a marker of cardiac pathology severity, were elevated in FGF[WT] but not FGF[Lo] mice compared to shams. Increased accumulation of the pro-cell death protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 was more pronounced in the FGF(WT) compared to FGF(Lo) mice, post TAC. Microarray analysis of the whole transcriptome of hearts in FGF2[WT] and FGF2[Lo] mice indicated the pathway linked to circadian rhythm as a candidate for the most significant differentially regulated. Specifically, upregulation of the circadian rhythm master regulator, Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1), was validated by qPCR and protein immunoblotting in FGF[Lo] mice versus downregulation of NR1D1in FGF[WT] mice post-TAC, when compared to their sham operated littermates. Taken together these studies suggest that expression of Hi-FGF2 contributes to cardiac systolic dysfunction in left ventricular pressure overloaded WT mice by downregulation of Nr1D1, post-TAC.

Cardiac muscle (the myocardium) is a unique arrangement of atria and ventricles that are spatially and electrically separated by a fibrous border. The spirally-arranged myocytes in both left and right ventricles are tethered by the component molecules of the cardiac extracellular matrix (ECM), including fibrillar collagen types I and III. Loss of normal arrangement of the ECM with either too little (as is observed in acute myocardial infarction) or too much (cardiac fibrosis in chronic post-myocardial infarction) is the primary contributor to cardiac dysfunction and heart failure. Matricellular proteins exist as non-structural signaling moieties in the ECM, and in the context of cardiac hypertrophy and heart failure, secreted 90 kDa periostin protein has attracted intense scrutiny during the past decade. Secreted periostin is now recognized for its important role in ECM development and maturation, as well as cellular adhesion. The novel mechanisms of periostin function include its role as a mediator of cell-to-matrix signaling, cell survival, and epithelial-mesenchymal transition (EMT). A number of recent studies have examined the hypothesis that periostin is a major contributor to ECM remodeling in the heart, and a number of very recent studies underscore its important role. This review examines recent developments in the mechanisms of periostin function in the normal heart and vasculature, and discusses recent advances which underpin its putative role in the development of cardiovascular disease. Periostin expression is very low at baseline in healthy tissues, but is re-expressed in damaged heart and in vessel walls after injury, in activated cardiac myofibroblasts and vascular smooth muscle cells, respectively. For this reason, periostin may be exploited for investigation of mechanisms of cardiac fibrosis, and we speculate that data generated from studies utilizing this approach may shed light on the timing for application of periostin-specific therapies to quell cardiac fibrosis and associated cardiac dysfunction.

Cardiovascular disease leading to heart failure (HF) remains a leading cause of morbidity and mortality worldwide. Improved pharmacological and interventional coronary procedures have led to improved outcomes following acute myocardial infarction. This success has translated into an unforeseen increased incidence in HF. This review summarizes the signaling pathways implicated in the transition to HF following cardiac injury. In addition, we provide an update on cell death signaling and discuss recent advances in cardiac fibrosis as an independent event leading to HF. Finally, we discuss cell-based therapies and their possible use to avert the deteriorating nature of HF. © 2019 American Physiological Society. Compr Physiol 9:75-125, 2019.

Following cardiac injury, fibroblasts are activated and are termed as myofibroblasts, and these cells are key players in extracellular matrix (ECM) remodeling and fibrosis, itself a primary contributor to heart failure. Nutraceuticals have been shown to blunt cardiac fibrosis in both in-vitro and in-vivo studies. However, nutraceuticals have had conflicting results in clinical trials, and there are no effective therapies currently available to specifically target cardiac fibrosis. We have previously shown that expression of the zinc finger E box-binding homeobox 2 (Zeb2) transcription factor increases as fibroblasts are activated. We now show that Zeb2 plays a critical role in fibroblast activation. Zeb2 overexpression in primary rat cardiac fibroblasts is associated with significantly increased expression of embryonic smooth muscle myosin heavy chain (SMemb), ED-A fibronectin and &alpha, smooth muscle actin (&alpha, SMA). We found that Zeb2 was highly expressed in activated myofibroblast nuclei but not in the nuclei of inactive fibroblasts. Moreover, ectopic Zeb2 expression in myofibroblasts resulted in a significantly less migratory phenotype with elevated contractility, which are characteristics of mature myofibroblasts. Knockdown of Zeb2 with siRNA in primary myofibroblasts did not alter the expression of myofibroblast markers, which may indicate that Zeb2 is functionally redundant with other profibrotic transcription factors. These findings add to our understanding of the contribution of Zeb2 to the mechanisms controlling cardiac fibroblast activation.

Primary cardiac fibroblasts are notoriously difficult to maintain for extended periods of time in cell culture, due to the plasticity of their phenotype and sensitivity to mechanical input. In order to study cardiac fibroblast activation in vitro, we have developed cell culture conditions which promote the quiescent fibroblast phenotype in primary cells. Using elastic silicone substrata, both rat and mouse primary cardiac fibroblasts could be maintained in a quiescent state for more than 3 days after isolation and these cells showed low expression of myofibroblast markers, including fibronectin extracellular domain A, non-muscle myosin IIB, platelet-derived growth factor receptor-alpha and alpha-smooth muscle actin. Gene expression was also more fibroblast-like vs. that of myofibroblasts, as Tcf21 was significantly upregulated, while Fn1-EDA, Col1A1 and Col1A2 were markedly downregulated. Cell culture conditions (eg. serum, nutrient concentration) are critical for the control of temporal fibroblast proliferation. We propose that eliminating mechanical stimulus and limiting the nutrient content of cell culture media can extend the quiescent nature of primary cardiac fibroblasts for physiological analyses in vitro.

Hearts donated following circulatory death (DCD) may represent an additional source of organs for transplantation; however, the impact of donor extubation on the DCD heart has not been well characterized. We sought to describe the physiologic changes that occur following withdrawal of life-sustaining therapy (WLST) in a porcine model of DCD. Physiologic changes were monitored continuously for 20 min following WLST. Ventricular pressure, volume, and function were recorded using a conductance catheter placed into the right (N = 8) and left (N = 8) ventricles, and using magnetic resonance imaging (MRI, N = 3). Hypoxic pulmonary vasoconstriction occurred following WLST, and was associated with distension of the right ventricle (RV) and reduced cardiac output. A 120-fold increase in epinephrine was subsequently observed that produced a transient hyperdynamic phase; however, progressive RV distension developed during this time. Circulatory arrest occurred 7.6±0.3 min following WLST, at which time MRI demonstrated an 18±7% increase in RV volume and a 12±9% decrease in left ventricular volume compared to baseline. We conclude that hypoxic pulmonary vasoconstriction and a profound catecholamine surge occur following WLST that result in distension of the RV. These changes have important implications on the resuscitation, preservation, and evaluation of DCD hearts prior to transplantation.

The resuscitation of hearts donated after circulatory death (DCD) is gaining widespread interest; however, the method of initial reperfusion (IR) that optimizes functional recovery has not been elucidated. We sought to determine the impact of IR temperature on the recovery of myocardial function during ex vivo heart perfusion (EVHP). Eighteen pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-min standoff period was observed and then hearts were reperfused for 3 min at three different temperatures (5°C; N = 6, 25°C; N = 5, and 35°C; N = 7) with a normokalemic adenosine-lidocaine crystalloid cardioplegia. Hearts then underwent normothermic EVHP for 6 h during which time myocardial function was assessed in a working mode. We found that IR coronary blood flow differed among treatment groups (5°C = 483 ± 53, 25°C = 722 ± 60, 35°C = 906 ± 36 mL/min, p

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.

Tissue development and homeostasis are dependent upon the concerted synthesis, maintenance, and degradation of extracellular matrix (ECM) molecules. Cardiac fibrosis is now recognized as a primary contributor to incidence of heart failure, particularly heart failure with preserved ejection fraction, wherein cardiac filling in diastole is compromised. Periostin is a cell-associated protein involved in cell fate determination, proliferation, tumorigenesis, and inflammatory responses. As a non-structural component of the ECM, secreted 90 kDa periostin is emerging as an important matricellular factor in cardiac mesenchymal tissue development. In addition, periostin's role as a mediator in cell-matrix crosstalk has also garnered attention for its association with fibroproliferative diseases in the myocardium, and for its association with TGF-β/BMP signaling. This review summarizes the phylogenetic history of periostin, its role in cardiac development, and the major signaling pathways influencing its expression in cardiovascular pathology. Further, we provide a synthesis of the current literature to distinguish the multiple roles of periostin in cardiac health, development and disease. As periostin may be targeted for therapeutic treatment of cardiac fibrosis, these insights may shed light on the putative timing for application of periostin-specific therapies.

Many etiologies of heart disease are characterized by expansion and remodeling of the cardiac extracellular matrix (ECM or matrix) which results in cardiac fibrosis. Cardiac fibrosis is mediated in cardiac fibroblasts by TGF‐β 1/R‐Smad2/3 signaling. Matrix component proteins are synthesized by activated resident cardiac fibroblasts known as myofibroblasts (MFB). These events are causal to heart failure with diastolic dysfunction and reduced cardiac filling. We have shown that exogenous Ski, a TGF‐β 1/Smad repressor, localizes in the cellular nucleus and deactivates cardiac myofibroblasts. This deactivation is associated with reduction of myofibroblast marker protein expression in vitro, including alpha smooth muscle actin (α‐SMA) and extracellular domain‐A (ED‐A) fibronectin. We hypothesize that Ski also acutely regulates MMP expression in cardiac MFB. While acute Ski overexpression in cardiac MFB in vitro was not associated with any change in intracellular MMP‐9 protein expression versus LacZ‐treated controls,exogenous Ski caused elevated MMP‐9 mRNA expression and increased MMP‐9 protein secretion versus controls. Zymographic analysis revealed increased MMP‐9‐specific gelatinase activity in myofibroblasts overexpressing Ski versus controls. Moreover, Ski expression was attended by reduced paxillin and focal adhesion kinase phosphorylation (FAK ‐ Tyr 397) versus controls. As myofibroblasts are hypersecretory and less motile relative to fibroblasts, Ski's reduction of paxillin and FAK expression may reflect the relative deactivation of myofibroblasts. Thus, in addition to its known antifibrotic effects, Ski overexpression elevates expression and extracellular secretion/release of MMP‐9 and thus may facilitate internal cytoskeletal remodeling as well as extracellular ECM components. Further, as acute TGF‐β 1 treatment of primary cardiac MFB is known to cause rapid translocation of Ski to the nucleus, our data support an autoregulatory role for Ski in mediating cardiac ECM accumulation.

// Shivika S. Gupta 1 , Matthew R. Zeglinski 1 , Sunil G. Rattan 1 , Natalie M. Landry 1 , Saeid Ghavami 2, 3 , Jeffrey T. Wigle 5 , Thomas Klonisch 2 , Andrew J. Halayko 3, 4 , Ian M.C. Dixon 1 1 Department of Physiology and Pathophysiology, Institute of Cardiovascular Sciences, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 2 Department of Human Anatomy and Cell Science, Basic Medical Sciences Building, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 3 Children’s Hospital Research Institute of Manitoba, John Buhler Research Centre, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 4 Department of Physiology and Pathophysiology, Internal Medicine and Pediatrics and Child Health, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada 5 Department of Biochemistry and Medical Genetics, Institute of Cardiovascular Sciences, Rady Faculty of Health Sciences, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada Correspondence to: Ian M.C. Dixon, email: idixon@sbrc.ca Keywords: cardiac fibroblast, myofibroblast, phenoconversion, autophagy, cardiac fibrosis Received: August 04, 2016 Accepted: September 20, 2016 Published: October 01, 2016 ABSTRACT The incidence of heart failure with concomitant cardiac fibrosis is very high in developed countries. Fibroblast activation in heart is causal to cardiac fibrosis as they convert to hypersynthetic cardiac myofibroblasts. There is no known treatment for cardiac fibrosis. Myofibroblasts contribute to the inappropriate remodeling of the myocardial interstitium, which leads to reduced cardiac function and ultimately heart failure. Elevated levels of autophagy have been linked to stress-induced ventricular remodeling and other cardiac diseases. Previously, we had shown that TGF-β 1 treatment of human atrial fibroblasts both induced autophagy and enhanced the fibrogenic response supporting a linkage between the myofibroblast phenotype and autophagy. We now demonstrate that with in vitro culture of primary rat cardiac fibroblasts, inhibition of autophagy represses fibroblast to myofibroblast phenoconversion. Culturing unpassaged cardiac fibroblasts for 72 hours on plastic tissue culture plates is associated with elevated α-smooth muscle actin (α-SMA) expression. This activation parallels increased microtubule-associated protein 1A/1B-light chain 3 (LC-3β II) protein expression. Inhibition of autophagy with bafilomycin-A1 (Baf-A1) and chloroquine (CQ) in cardiac fibroblasts significantly reduces α-SMA and extracellular domain A fibronectin (ED-A FN) protein vs untreated controls. Myofibroblast cell migration and contractility were significantly reduced following inhibition of autophagy. These data support the possibility of a causal link between cardiac fibroblast-to-myofibroblast phenoconversion and autophagy.

Background: Hearts donated after circulatory death may represent an additional donor source. The influx of sodium and calcium ions across the sarcolemma play a central role in the pathogenesis of ischemia-reperfusion injury; however, this process may be inhibited if the initial reperfusion solution is rendered hypocalcemic and acidic. We sought to determine the calcium concentration and pH of the initial reperfusion solution that yielded optimal functional recovery of hearts donated after circulatory death during ex vivo heart perfusion. Methods: Pigs were anesthetized, mechanical ventilation was discontinued, and a 15-minute standoff period was observed after circulatory arrest. Hearts were reperfused with a normothermic cardioplegia of varying calcium concentrations (part 1 [50 μmol/L, n = 4; 220 μmol/L, n = 9; 500 μmol/L, n = 4; and 1,250 μmol/L, n = 5]) and pH (part 2 [7.9, n = 5; 7.4, n = 9; 6.9, n = 8; and 6.4, n = 6]). Myocardial function was then assessed in a physiologic working model 1 hour after initiation of normothermic ex vivo heart perfusion. Results: The calcium concentration and pH of the cardioplegic solution affected the development of myocardial edema (part 1: 50 μmol/L = 5.8% ± 0.9%; 220 μmol/L = 4.3% ± 0.4%; 500 μmol/L = 7.0% ± 0.6%; and 1,250 μmol/L = 6.6% ± 0.8% weight gain, p = 0.015; part 2: 7.9 = 3.6% ± 0.4%, 7.4 = 4.3% ± 0.4%, 6.9 = 3.7% ± 0.6%, and 6.4 = 6.4% ± 1.3% weight gain, p = 0.056) and the recovery of myocardial function (cardiac index part 1: 50 μmol/L = 2.6 ± 0.6; 220 μmol/L = 6.0 ± 0.8; 500 μmol/L = 2.3 ± 0.5; and 1,250 μmol/L = 1.9 ± 0.6 mL · m−1 · g−1, p < 0.001; part 2: 7.9 = 1.5 ± 0.7; 7.4 = 6.0 ± 0.8; 6.9 = 8.4 ± 1.8; and 6.4 = 3.1 ± 0.8 mL · m−1 · g−1, p = 0.003) during ex vivo heart perfusion. Conclusions: Initial reperfusion of hearts donated after circulatory death with a hypocalcemic and moderately acidic cardioplegia minimizes edema and optimizes functional recovery during subsequent ex vivo heart perfusion.

Inappropriate cardiac interstitial remodeling is mediated by activated phenoconverted myofibroblasts. The synthesis of matrix proteins by these cells is triggered by both chemical and mechanical stimuli. Ski is a repressor of TGFβ1/Smad signaling and has been described as possessing anti-fibrotic properties within the myocardium. We hypothesized that overexpression of Ski in myofibroblasts will induce an apoptotic response, which may either be supported or opposed by autophagic flux. We used primary myofibroblasts (activated fibroblasts) which were sourced from whole heart preparations that were only passaged once. We found that overexpression of Ski results in distinct morphological and biochemical changes within primary cardiac myofibroblasts associated with apoptosis. Ski treatment was associated with the expression of pro-apoptotic factors such as Bax, caspase-7, and -9. Our results indicate that Ski triggers a pro-death mechanism in primary rat cardiac myofibroblasts that is mediated through the intrinsic apoptotic pathway. Myofibroblast survival is prolonged by an autophagic response, as the dataset indicate that apoptosis is hastened when autophagy is inhibited. We suggest that the apoptotic death response of myofibroblasts is working in parallel with the previously observed anti-fibrotic properties of Ski within this cell type. As myofibroblasts are the sole mediators of matrix expansion in heart failure, we suggest that Ski, or a putative Ski-mimetic, may induce graded apoptosis in myofibroblasts within the failing heart and may be a novel therapeutic approach towards controlling cardiac fibrosis. Future studies are needed to examine the potential effects of Ski overexpression on other cell types in the heart.

Chemotactic movement of myofibroblasts is recognized as a common means for their sequestration to the site of tissue injury. Following myocardial infarction (MI), recruitment of cardiac myofibroblasts to the infarct scar is a critical step in wound healing. Contractile myofibroblasts express embryonic smooth muscle myosin, α-smooth muscle actin, as well as collagens I and III. We examined the effects of cardiotrophin-1 (CT-1) in the induction of primary rat ventricular myofibroblast motility. Changes in membrane potential (Em) and Ca2+entry were studied to reveal the mechanisms for induction of myofibroblast migration. CT-1-induced cardiac myofibroblast cell migration, which was attenuated through the inhibition of JAK2 (25 μM AG490), and myosin light chain kinase (20 μM ML-7). Inhibition of K+channels (1 mM tetraethylammonium or 100 μM 4-aminopyridine) and nonselective cation channels by 10 μM gadolinium (Gd3+) significantly reduced migration in the presence of CT-1. CT-1 treatment caused a significant increase in myosin light chain phosphorylation, which could be inhibited by incubation in Ca2+-free conditions or by application of AG490, ML-7, and W7 (100 μM; calmodulin inhibitor). Monitoring myofibroblast membrane potential with potentiometric fluorescent DiBAC4( 3 ) dye revealed a biphasic response to CT-1 consisting of an initial depolarization followed by hyperpolarization. Increased intracellular Ca2+, as assessed by fluo 3, occurred immediately after membrane depolarization and attenuated at the time of maximal hyperpolarization. CT-1 exerts chemotactic effects via multiple parallel signaling modalities in ventricular myofibroblasts, including changes in membrane potential, alterations in intracellular calcium, and activation of a number of intracellular signaling pathways. Further study is warranted to determine the precise role of K+currents in this process.

A high-lipid diet (HLD) may lead to adverse left ventricular (LV) remodeling and endothelial dysfunction in conditions of hemodynamic stress. Although congenital absence of nitric oxide synthase 3 (NOS3) leads to adverse LV remodeling after transverse aortic constriction (TAC), the effects of a HLD in this state remains unknown. Wild-type (WT) and NOS3 knockout mice (NOS3 -/- ) were randomized into the following 4 groups: 1) WT + low-lipid diet (LLD) (10% of energy); 2) WT + HLD (60% of energy); 3) NOS3 -/- + LLD; and 4) NOS3 -/- + HLD for a total of 12 wk. After 1 wk of randomization, TAC was performed on all groups. Serial echocardiography revealed a decrease in LV ejection fraction (LVEF) in WT and NOS3 -/- mice fed the HLD compared with those fed the LLD diet at 12 wk post-TAC. Mice fed the NOS3 -/- + HLD diet had a lower LVEF compared with mice in the other 3 groups (P < 0.05). There was greater myocyte hypertrophy, interstitial fibrosis, and percentage change in plasma cholesterol concentrations in the NOS3 -/- + HLD group 12 wk post-TAC compared with the other 3 groups. Although high molecular weight fibroblast growth factor-2, a marker of cardiac hypertrophy, was more upregulated in the NOS3 -/- + HLD group than in the other groups, markers of the renin-angiotensin system did not differ among them. A HLD potentiates LV dysfunction in NOS3 -/- mice in a chronic pressure overload state.

Recent studies demonstrate the critical role of the extracellular matrix in the organization of parenchymal cells in the heart. Thus, an understanding of the modes of regulation of matrix production by cardiac myofibroblasts is essential. Transforming growth factor β (TGF-β) signaling is transduced through the canonical Smad pathway, and the involvement of this pathway in matrix synthesis and other processes requires precise control. Inhibition of Smad signaling may be achieved at the receptor level through the targeting of the TGF-β type I receptors with an inhibitory Smad7 / Smurf2 complex, or at the transcriptional level through c-Ski / receptor-Smad / co-mediator Smad4 interactions. Conversely, Arkadia protein intensifies TGF-β-induced effects by marking c-Ski and inhibitory Smad7 for destruction. The study of these TGF-β mediators is essential for future treatment of fibrotic disease, and this review highlights recent relevant findings that may impact our understanding of cardiac fibrosis.

The transcription factor scleraxis has been implicated in regulating the development of collagen-rich tissues such as tendons and cardiac valves, but its role in general collagen synthesis in the heart is unknown. Scleraxis expression in cardiac fibroblasts was examined, and its ability to regulate gene expression of collagen I alpha 2, the predominant cardiac collagen isoform, was assayed. Using real-time PCR, we demonstrate here that scleraxis mRNA is up-regulated by the profibrotic agonist TGF-beta(1) in rat cardiac myofibroblasts, and that phenoconversion of fibroblasts to myofibroblasts similarly increases scleraxis expression. Over-expression of scleraxis in NIH-3T3 or primary rat cardiac fibroblasts by adenoviral gene delivery is sufficient to significantly increase collagen I alpha 2 gene expression. Using luciferase reporter assays, we demonstrate that scleraxis transactivates the human collagen I alpha 2 promoter in a DNA- and protein-binding dependent manner. Intriguingly, examination of infarcted rat hearts reveals a nearly four-fold increase in scleraxis expression in the infarct scar, but not in non-infarcted tissue. These data support a novel and previously unknown role for scleraxis in the regulation of collagen gene expression in the heart, including in post-infarct scar formation.

Myocardial matrix remodeling is a well-recognized disease modifier in the pathogenesis of heart failure, although the precise underlying molecular mechanisms remain to be elucidated. Here we investigated the effects of leptin, circulating levels of which are typically increased in obese individuals, on MMP and collagen expression and MMP activity in isolated cardiac myofibroblasts. Neonatal rat myofibroblasts were treated with 6 nM recombinant leptin and the collected supernatant analyzed for MMP-2 activity via gelatin zymography. MMP-2, MT1-MMP and procollagen-I and -III protein expression were determined by western blotting and MMP-2 and MT1-MMP mRNA expression were examined utilizing real-time PCR. Procollagen-I levels were analyzed by confocal microscopy and collagen synthesis was determined through [ 3 H]-proline incorporation. Exposure of myofibroblasts to leptin (24 h) significantly increased MMP-2 activity, while mRNA and protein levels remained unchanged. Leptin also significantly enhanced mRNA and protein expression of MT1-MMP, a known activator of MMP-2. Biotinylation assays indicated increased cell surface expression of MT1-MMP in response to leptin and use of a MT1-MMP inhibitor attenuated the leptin-mediated elevation of MMP-2 activity. Total cellular collagen synthesis was unaffected by leptin treatment, however intracellular procollagen-I protein was significantly increased in treated cells. Furthermore, extracellular soluble procollagen-I was increased, while a decrease in soluble procollagen-III protein was observed in conditioned media. In summary, these findings in isolated cardiac myofibroblasts support the suggestion that leptin may directly influence myocardial matrix metabolism, and this may represent a mechanism contributing to cardiac fibrosis in obese patients with elevated plasma leptin levels.

Cardiac ventricular myofibroblast motility, proliferation, and contraction contribute to post-myocardial infarct wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The Na+-Ca2+ exchanger (NCX1) is involved in altered calcium handling in cardiac myocytes during cardiac remodeling associated with heart failure, however, its role in cardiac myofibroblast cell function is unexplored. In this study we investigated the involvement of NCX1 as well as the role of non-selective-cation channels (NSCC) in cardiac myofibroblast cell function in vitro. Immunofluorescence and Western blots revealed that P1 cells upregulate alpha-smooth muscle actin (alphaSMA) and embryonic smooth muscle myosin heavy chain (SMemb) expression. NCX1 mRNA and proteins as well as Ca(v)1.2a protein are also expressed in P1 myofibroblasts. Myofibroblast motility in the presence of 50 ng/ml PDGF-BB was blocked with AG1296. Myofibroblast motility, contraction, and proliferation were sensitive to KB-R7943, a specific NCX1 reverse-mode inhibitor. In contrast, only proliferation and contraction, but not motility were sensitive to nifedipine, while gadolinium (NSCC blocker) was only associated with decreased motility. ML-7 treatment was associated with inhibition of the chemotactic response and contraction. Thus cardiac myofibroblast chemotaxis, contraction, and proliferation were sensitive to different pharmacologic treatments suggesting that regulation of transplasmalemmal calcium movements may be important in growth factor receptor-mediated processes. NCX1 may represent an important moiety in suppression of myofibroblast functions.

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

Ex vivo heart perfusion (EVHP) may facilitate resuscitation of discarded donor hearts and expand the donor pool; however, a reliable means of demonstrating organ viability prior to transplantation is required. Therefore, we sought to identify metabolic and functional parameters that predict myocardial performance during EVHP. To evaluate the parameters over a broad spectrum of organ function, we obtained hearts from 9 normal pigs and 37 donation after circulatory death pigs and perfused them ex vivo. Functional parameters obtained from a left ventricular conductance catheter, oxygen consumption, coronary vascular resistance, and lactate concentration were measured, and linear regression analyses were performed to identify which parameters best correlated with myocardial performance (cardiac index: mL·min–1·g–1). Functional parameters exhibited excellent correlation with myocardial performance and demonstrated high sensitivity and specificity for identifying hearts at risk of poor post-transplant function (ejection fraction: R2 = 0.80, sensitivity = 1.00, specificity = 0.85; stroke work: R2 = 0.76, sensitivity = 1.00, specificity = 0.77; minimum dP/dt: R2 = 0.74, sensitivity = 1.00, specificity = 0.54; tau: R2 = 0.51, sensitivity = 1.00, specificity = 0.92), whereas metabolic parameters were limited in their ability to predict myocardial performance (oxygen consumption: R2 = 0.28; coronary vascular resistance: R2 = 0.20; lactate concentration: R2 = 0.02). We concluded that evaluation of functional parameters provides the best assessment of myocardial performance during EVHP, which highlights the need for an EVHP device capable of assessing the donor heart in a physiologic working mode.

Cardiac fibroblast to myofibroblast phenoconversion is a critical step in cardiovascular disease progression. Myofibroblasts contribute to cardiac fibrosis by chronic extracellular matrix remodelli...

Cardiac fibrosis is the pathological accumulation of cardiac extracellular matrix (ECM or matrix), which occurs in most types of heart disease. Major recent advances in our understanding have allowed us to identify cardiac fibrosis as a primary disease independent of either cardiomyocyte injury or loss. New developments within this field are burgeoning, including research that points to multiple sources for cardiac myofibroblasts participating in cardiovascular disease pathogenesis, the feasibility of bioengineered matrix tissues as well as the identification of novel targets to reduce the incidence and severity of cardiac fibrosis. A summary of the state of knowledge of the regulation of the function of fibroblasts as well as a synopsis of the current state of investigation to address the biology of cardiovascular fibroblasts, valvular interstitial cells (VICs), and myofibroblasts is warranted. This book will help to adapt the information that we have gathered in order to translate it into treatments for fibrotic cardiac diseases and thus alter the course of their progression.

“Cardiovascular disease (CVD) is a growing epidemic and the leading cause of mortality worldwide. More than one in three Americans are living with some form of CVD. Despite the vast diversity of CVD forms, many disease states are associated with maladaptive remodeling of the myocardial interstitium. Elevated fibrillar collagen expression is considered to be the primary contributor to altered cardiac function based on its adverse influence on electrical signal transduction, myocardial stiffness, and cardiac dysfunction. Amongst the various mechanisms responsible for the production of collagen fibres, transforming growth factor-β1 (TGF-β1) has been identified as a critical mediator of the fibrotic response in the injured myocardium. Herein we describe a potential role for the TGF-β1 negative regulators Ski/Sno, and the TGF-β1 transcriptional regulators YAP/TAZ in cardiac fibroblast and myofibroblast phenotype and function through modulation of TGF-β1 signaling.”

Remodeling of the extracellular matrix is beneficial during the acute wound healing stage following tissue injury. In the short term, resident fibroblasts and myofibroblasts regulate the matrix remodeling process through production of matricellular protein components that provide structural support to the damaged tissue. This process is largely governed by the transforming growth factor-β1(TGF-β1) pathway, a critical mediator of the remodeling process. In the long term, chronic activation of the TGF-β1pathway promotes excessive synthesis and deposition of matrix proteins, including fibrillar collagens, which ultimately leads to organ failure. SnoN (and its alternatively-spliced isoforms SnoN2, SnoA, and SnoI) is one of four members of a family of negative regulators of TGF-β1signaling that includes Ski and functional Smad-suppressing elements on chromosomes 15 and 18. SnoN has been shown to be structurally and functionally similar to Ski and has been demonstrated to directly interact with Ski to abrogate gene expression. Despite this, little progress has been made in delineating a specific role for SnoN in the regulation of myofibroblast phenotype and function. This review outlines the current body of knowledge of what we refer to as the “Ski-Sno superfamily,” with a focus on the structural and functional importance of SnoN in mediating the fibrotic response by myofibroblasts following tissue injury.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K+ currents were recorded at depolarized potentials, and an inwardly rectifying K+ (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K+ concentration ([K+]o) was raised, and this Kir current was blocked by 100–300 μM Ba2+. RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K+]o influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC3(4), showed that 1.5 mM [K+]o resulted in a hyperpolarization, whereas 20 mM [K+]o produced a depolarization. Low [K+]o (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K+]o). In contrast, 20 mM [K+]o resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K+]o significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K+]o. In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K+ channel α-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Ischemic heart disease is the most common cause of mortality worldwide. Cardiac fibroblasts and myofibroblasts, i.e., the hypersecretory, muscular, and contractile fibroblastic phenotype variant, play an important role in myocardial healing and are responsible for accumulation of collagen in the infarct scar as well as in viable myocardium. Thus, cardiac fibroblasts and myofibroblasts directly contribute to cardiac stiffness, altered performance, and ultimately to the onset of systolic and diastolic heart failure. Cardiotrophin-1 (CT-1) is a member of the IL-6 superfamily and is elevated in the serum of patients with ischemic heart disease and valvular heart disease; it is also known to induce cardiomyocyte hypertrophy in vitro. The recent, burgeoning awareness of the functions of IL-6 superfamily of cytokines within cardiovascular diseases predicates this summary of CT-1's effect in cardiac wound healing, and particularly after the induction of myocardial infarction. Further, we summarize recent results of cardiac CT-1 expression post-myocardial infarction (post-MI) as well as the effect of CT-1 on cultured primary adult rat cardiac fibroblasts with respect to proliferation and collagen secretion. It would appear that CT-1 plays an important and heretofore largely unrecognized role in infarct scar formation and angiogenesis in the rat model of chronic MI. Further work is required to determine factors that induce CT-1 expression, its interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure, and ultimately whether it confers a beneficial effect or contributes to maladaptive cardiac fibrosis.

Objective: Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines and is expressed in various cardiovascular disease states. CT-1 induces cardiomyocyte hypertrophy, and protects myocytes from ischemia reperfusion injury. We sought to elucidate CT-1 signaling in cardiac fibroblasts with respect to initiation of protein synthesis. Methods: Cardiac fibroblasts were isolated from the ventricles of 200-g Sprague–Dawley rats and stimulated with CT-1 at specified concentrations with or without inhibitors of cell signaling pathways. Activation of intracellular signaling pathways was determined by Western analysis and immunocytochemistry. Protein synthesis was measured by incorporation of [3H]leucine. Results: CT-1 treatment resulted in activation of the Jak/STAT, MAPK, and Akt pathways in addition to protein synthesis regulatory proteins with resultant increase in overall protein synthesis. Analysis with phospho-specific antibodies revealed that AG490 (Jak inhibitor), PD98059 (MEK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), LY294002 (PI3-K inhibitor) and rapamycin (mTOR inhibitor) act at different levels in the signaling cascade to inhibit CT-1 induced protein synthesis. Conclusion: Cardiotrophin-1 activates the Jak/STAT, PI3K/Akt, p38 and p42/44 MAPK pathways in cardiac fibroblasts. Use of pharmacologic inhibitors reveals that each of these pathways play a role in CT-1 induced protein synthesis.

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT1 receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000–1800 cm−1 region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-β strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT1 blockade is associated with attenuation of collagen absorption in these lesions.

Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been shown to be elevated in the serum of patients with ischemic heart disease and valvular heart disease, and induces cardiomyocyte hypertrophy in vitro. We investigated expression of CT-1 in post-MI rat heart and the effect of CT-1 on cultured primary adult rat cardiac fibroblasts. Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of Jak/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased 3H-leucine and 3H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (PICP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. Our results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis. Further work is required to determine factors that induce CT-1 expression and interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure.

See related article, pp 264–270 The term “fibroblast” designates a highly heterogenous group of cells that exhibit distinct differentiated phenotypes in different organs.1 The study of fibroblast and myofibroblast biology in specific organs is an important but relatively understudied area, especially in the heart. Recent novel data indicate that ventricular fibroblast activation and cardiac fibrosis are primary events in ventricular remodeling rather than secondary to cardiomyocyte injury.2 In larger mammalian species, including humans, cardiac fibroblasts represent the most numerous nonmyocytes in the myocardium. These cells function to synthesize and organize collagens, fibronectins, and other interstitial components and, thus, maintain the integrity of the cardiac extracellular matrix (matrix). Matrix remodeling can manifest as interstitial fibrosis of an otherwise normal myocardium. This remodeling may occur with the onset of hypertension or as the progressive evolution of the structure of the infarct scar. Remodeling of the matrix occurs later in the noninfarcted myocardium, in the etiology of heart failure after myocardial infarction. The dogma that cardiac fibrosis is merely a secondary disease modifier after cardiomyocyte damage is losing ground to the idea that fibrosis is a primary disease prima facie. Therefore, the need to identify and characterize the specific signals that might trigger the phenoconversion of relatively quiescent fibroblasts to myofibroblasts takes on new importance. In normal heart tissue, matrix protein secretion and deposition are carried out exclusively by cardiac fibroblasts with relatively low turnover of proteins. Conversely, contractile and hypersynthetic myofibroblasts are the relevant phenotypic variants in wound healing or in hypertrophied and …

The fibroblast to myofibroblast phenoconversion, which is induced by TGF-β, is a crucial step during cardiac fibrosis. Activated myofibroblasts increase extracellular matrix (ECM) synthesis, which ...

Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-β1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis. © 2014 Springer Science+Business Media New York.

Rapid progress made in various areas of regenerative medicine in recent years occurred both at the cellular level, with the Nobel prize-winning discovery of reprogramming (generation of induced pluripotent stem (iPS) cells) and also at the biomaterial level. The use of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4 (called commonly "Yamanaka factors") for the conversion of differentiated cells, back to the pluripotent/embryonic stage, has opened virtually endless and ethically acceptable source of stem cells for medical use. Various types of stem cells are becoming increasingly popular as starting components for the development of replacement tissues, or artificial organs. Interestingly, many of the transcription factors, key to the maintenance of stemness phenotype in various cells, are also overexpressed in cancer (stem) cells, and some of them may find the use as prognostic factors. In this review, we describe various methods of iPS creation, followed by overview of factors known to interfere with the efficiency of reprogramming. Next, we discuss similarities between cancer stem cells and various stem cell types. Final paragraphs are dedicated to interaction of biomaterials with tissues, various adverse reactions generated as a result of such interactions, and measures available, that allow for mitigation of such negative effects.

Background . After acute myocardial infarction, regional myocardial wall strains and stresses change and a complex cellular and biochemical response is initiated to remodel the ventricle. This study tests the hypothesis that changes in regional ventricular wall strains affect regional collagen accumulation and collagenase activity. Methods . Fourteen sheep had acute anteroapical infarction that progressively expands into left ventricular aneurysm within 8 weeks. In 7 sheep, infarct expansion was restrained by prior placement of mesh over the area at risk. Fourteen days after infarction, and after hemodynamic and echocardiographic measurements, animals were euthanized for histology, measurements of hydroxyproline, matrix metalloproteinase-1 (MMP-1 or collagenase) and MMP-2 (gelatinase) activity, as well as collagen type I and III in infarcted, borderzone, and remote myocardium. Results . Restraining infarct expansion does not change collagen content or MMP-1 or MMP-2 activity in the infarct, but significantly increases the ratio of collagen I/III. In borderzone and remote myocardium infarct, restraint significantly increases collagen content and significantly reduces MMP-1 activity. MMP-2 activity is reduced ( p = 0.059) in borderzone myocardium only. Between groups, the ratio of type I/III fibrillar collagen does not change in borderzone myocardium. Conclusions . Fourteen days after acute myocardial infarction, restraining infarct expansion increases collagen accumulation in borderzone and remote myocardium, which may prevent expansion of hypocontractile, fully perfused "remodeling myocardium" adjacent to the infarct. This study demonstrates that changes in regional myocardial wall strain alter the cellular and biochemical processes involved in postinfarction ventricular remodeling.