Micronized Acellular Matrix Biomaterial Leverages Eosinophils for Postinfarct Cardiac Repair.

After ischemic injury, immune cells mediate maladaptive cardiac remodeling. Extracellular matrix biomaterials may redirect inflammation toward repair. Pericardial fluid contains pro-reparative immune cells, potentially leverageable by biomaterials. Herein, we explore how pericardial delivery of a micronized extracellular matrix biomaterial affects cardiac healing. In noninfarcted mice, pericardial delivery increases pericardial and myocardial eosinophil counts. This response is sustained after myocardial infarction, stimulating an interleukin 4 rich milieu. Ultimately, the biomaterial improves postinfarct vascularization and cardiac function; and eosinophil-knockout negates these benefits. For the first time, to our knowledge, we demonstrate the therapeutic potential of pericardial biomaterial delivery and the eosinophil's critical role in biomaterial-mediated postinfarct repair.

Acellular biomaterial modulates myocardial inflammation and promotes endogenous mechanisms of postinfarct cardiac repair.

OBJECTIVE: After myocardial infarction, we previously showed that epicardial implantation of porcine small intestinal submucosal extracellular matrix (SIS-ECM) improves postinfarct cardiac function through fibroblast-mediated angiogenic and antifibrotic pathways. Herein, we characterize how SIS-ECM also coordinates a reparative cardiac inflammatory response. METHODS: RNA sequencing and multiplex characterized modulation of fibroblast transcriptional and paracrine activity by SIS-ECM. Inhibitors of fibroblast growth factor 2 and toll-like receptor 9 elucidated mechanism. Mice received coronary ligation (infarction) and either SIS-ECM implantation (treatment) or sham surgery (control). Flow cytometry of SIS-ECM and the murine myocardium quantified monocytes, neutrophils, and proangiogenic subtypes. Microscopy tracked fibroblasts and immune cells, and characterized myocardial angiogenesis. RESULTS: SIS-ECM increased fibroblast transcription of inflammatory pathways and production of angiogenic vascular endothelial growth factor and inflammatory cytokines via fibroblast growth factor 2 and toll-like receptor 9-dependent pathways. Two-photon microscopy showed that SIS-ECM became engrafted by native fibroblasts and leukocytes, subsequently increasing release of inflammatory cytokines and angiogenic vascular endothelial growth factor. On flow cytometry, SIS-ECM implantation increased day-7 myocardial counts of neutrophils, inflammatory monocytes, and proangiogenic vascular endothelial growth factor recptor 1 subtypes. SIS-ECM has a higher proportion of proangiogenic leukocytes compared with the myocardium. Resonant confocal microscopy showed neovascularization near SIS-ECM. CONCLUSIONS: SIS-ECM promotes engraftment by native fibroblasts and leukocytes, and modulates fibroblast activity via fibroblast growth factor 2 and toll-like receptor 9 to potentiate a proangiogenic inflammatory response. Subsequently, the material increases myocardial counts of reparative proangiogenic leukocytes that can induce neovascularization. This reparative inflammatory response may explain previously reported functional improvements. Fibroblast growth factor 2 and toll-like receptor 9 mechanisms can be leveraged to design next-generation materials for postinfarct cardiac repair.

Dye-Mediated Photo-Oxidation Biomaterial Fixation: Analysis of Bioinductivity and Mechanical Properties of Bovine Pericardium for Use in Cardiac Surgery.

Extracellular matrix bioscaffolds can influence the cardiac microenvironment and modulate endogenous cellular mechanisms. These materials can optimize cardiac surgery for repair and reconstruction. We investigated the biocompatibility and bioinductivity of bovine pericardium fixed via dye-mediated photo-oxidation on human cardiac fibroblast activity. We compared a dye-mediated photo-oxidation fixed bioscaffold to glutaraldehyde-fixed and non-fixed bioscaffolds reported in contemporary literature in cardiac surgery. Human cardiac fibroblasts from consenting patients were seeded on to bioscaffold materials to assess the biocompatibility and bioinductivity. Human cardiac fibroblast gene expression, secretome, morphology and viability were studied. Dye-mediated photo-oxidation fixed acellular bovine pericardium preserves human cardiac fibroblast phenotype and viability; and potentiates a pro-vasculogenic paracrine response. Material tensile properties were compared with biomechanical testing. Dye-mediated photo-oxidation fixed acellular bovine pericardium had higher compliance compared to glutaraldehyde-fixed bioscaffold in response to tensile force. The biocompatibility, bioinductivity, and biomechanical properties of dye-mediated photo-oxidation fixed bovine pericardium demonstrate its feasibility as a bioscaffold for use in cardiac surgery. As a fixed yet bioinductive solution, this bioscaffold demonstrates enhanced compliance and retains bioinductive properties that may leverage endogenous reparative pathways. Dye-mediated photo-oxidation fixed bioscaffold warrants further investigation as a viable tool for cardiac repair and reconstruction.

An Intact Pericardium Ischemic Rodent Model.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart's resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

Acellular bioscaffolds redirect cardiac fibroblasts and promote functional tissue repair in rodents and humans with myocardial injury.

Coronary heart disease is a leading cause of death. Tissue remodeling and fibrosis results in cardiac pump dysfunction and ischemic heart failure. Cardiac fibroblasts may rebuild damaged tissues when prompted by suitable environmental cues. Here, we use acellular biologic extracellular matrix scaffolds (bioscaffolds) to stimulate pathways of muscle repair and restore tissue function. We show that acellular bioscaffolds with bioinductive properties can redirect cardiac fibroblasts to rebuild microvascular networks and avoid tissue fibrosis. Specifically, when human cardiac fibroblasts are combined with bioactive scaffolds, gene expression is upregulated and paracrine mediators are released that promote vasculogenesis and prevent scarring. We assess these properties in rodents with myocardial infarction and observe bioscaffolds to redirect fibroblasts, reduce tissue fibrosis and prevent maladaptive structural remodeling. Our preclinical data confirms that acellular bioscaffold therapy provides an appropriate microenvironment to stimulate pathways of functional repair. We translate our observations to patients with coronary heart disease by conducting a first-in-human observational cohort study. We show that bioscaffold therapy is associated with improved perfusion of infarcted myocardium, reduced myocardial scar burden, and reverse structural remodeling. We establish that clinical use of acellular bioscaffolds is feasible and offers a new frontier to enhance surgical revascularization of ischemic heart muscle.

Direct Effects of Empagliflozin on Extracellular Matrix Remodelling in Human Cardiac Myofibroblasts: Novel Translational Clues to Explain EMPA-REG OUTCOME Results.

BACKGROUND: Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. METHODS: Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. RESULTS: Empagliflozin significantly attenuated transforming growth factor ß1-induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 µmol/L, 1 µmol/L, and 5 µmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. CONCLUSIONS: We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

Gata6+ Pericardial Cavity Macrophages Relocate to the Injured Heart and Prevent Cardiac Fibrosis.

Macrophages play an important role in structural cardiac remodeling and the transition to heart failure following myocardial infarction (MI). Previous research has focused on the impact of blood-derived monocytes on cardiac repair. Here we examined the contribution of resident cavity macrophages located in the pericardial space adjacent to the site of injury. We found that disruption of the pericardial cavity accelerated maladaptive post-MI cardiac remodeling. Gata6+ macrophages in mouse pericardial fluid contributed to the reparative immune response. Following experimental MI, these macrophages invaded the epicardium and lost Gata6 expression but continued to perform anti-fibrotic functions. Loss of this specialized macrophage population enhanced interstitial fibrosis after ischemic injury. Gata6+ macrophages were present in human pericardial fluid, supporting the notion that this reparative function is relevant in human disease. Our findings uncover an immune cardioprotective role for the pericardial tissue compartment and argue for the reevaluation of surgical procedures that remove the pericardium.

Induction of human aortic myofibroblast-mediated extracellular matrix dysregulation: A potential mechanism of fluoroquinolone-associated aortopathy.

OBJECTIVES: Fluoroquinolone (FQ) antibiotics are associated with adverse aortic clinical events. We assessed human aortic myofibroblast-mediated extracellular matrix (ECM) dysregulation as a possible cellular mechanism underlying FQ-associated aortopathy. METHODS: Human aortic myofibroblasts were isolated from patients with aortopathy undergoing elective ascending aortic resection (N = 9). The capacity for extracellular matrix degradation in cells exposed to FQ was assessed by multiplex analysis of secreted matrix metalloproteinases relative to tissue inhibitors of matrix metalloproteinases (TIMPs). Direct evaluation of extracellular matrix degradation was investigated in human aortic cells using a 3-dimensional gelatin-fluorescein isothiocyanate fluorescence microgel assay. Aortic cellular collagen-1 expression following FQ exposure was determined by immunoblotting and immunofluorescent staining. Cell apoptosis, necrosis, and metabolic viability was determined by annexin-V, propidium iodide staining, and water-soluble tetrazolium salt (WST1) assay. RESULTS: FQ exposure significantly decreased aortic cell TIMP-1 (P = .004) and TIMP-2 (P = .0004) protein expression compared with vehicle control. The ratio of matrix metalloproteinase-9/TIMP-2 was increased suggesting an increased capacity for extracellular matrix degradation (P = .01). In collagen gels, we show a trend toward increased aortic myofibroblast-mediated collagen fiber degradation with FQ exposure (P = .09). Similarly, FQ exposure attenuated collagen-1 expression as assessed by immunoblotting (P = .002) and immunofluorescence (P = .02). Cell apoptosis, necrosis, and metabolic viability was not significantly influenced by FQ exposure. CONCLUSIONS: For the first time, we document a putative mechanism underlying FQ-associated aortopathy whereby decreased TIMP expression with impaired compensatory collagen-1 expression results in human aortic myofibroblast-mediated extracellular matrix dysregulation. These novel data may provide a cellular and molecular mechanism to explain the established clinical association between FQ exposure and acute aortic events.

Human pericardial proteoglycan 4 (lubricin): Implications for postcardiotomy intrathoracic adhesion formation.

OBJECTIVE: Intrapericardial fibrous adhesions increase the risk of sternal reentry. Proteoglycan 4/lubricin (PRG4) is a mucin-like glycoprotein that lubricates tissue compartments and prevents inflammation. We characterized PRG4 expression in human pericardium and examined its effects in vitro on human cardiac myofibroblast fibrotic activity and in vivo as a measure of its therapeutic potential to prevent adhesions. METHODS: Full-length PRG4 expression was determined using Western blot analysis and amplified luminescent proximity homogeneous assay in human pericardial tissues obtained at cardiotomy. The in vitro effects of PRG4 were investigated on human cardiac myofibroblasts for cell adhesion, collagen gel contraction, and cell-mediated extracellular matrix remodeling. The influence of PRG4 on pericardial homeostasis was determined in a chronic porcine animal model. RESULTS: PRG4 is expressed in human pericardial fluid and colocalized with pericardial mesothelial cells. Recombinant human PRG4 prevented human cardiac myofibroblast attachment and reduced myofibroblast activity assessed using collagen gel contraction assay (64.6% ± 8.1% vs 47.1% ± 6.8%; P = .02). Using a microgel assay, human cardiac myofibroblast mediated collagen fiber remodeling was attenuated by PRG4 (1.17 ± 0.03 vs 0.90 ± 0.05; P = .002). In vivo, removal of pericardial fluid alone induced severe intrapericardial adhesion formation, tissue thickening, and inflammatory fluid collections. Restoration of intrapericardial PRG4 was protective against fibrous adhesions and preserved the pericardial space. CONCLUSIONS: For the first time, we show that PRG4 is expressed in human pericardial fluid and regulates local fibrotic myofibroblast activity. Loss of PRG4-enriched pericardial fluid after cardiotomy might induce adhesion formation. Therapeutic restoration of intrapericardial PRG4 might prevent fibrous/inflammatory adhesions and reduce the risk of sternal reentry.

Heparin Augmentation Enhances Bioactive Properties of Acellular Extracellular Matrix Scaffold.

Extracellular matrix (ECM) maintains a reservoir of bioactive growth factors and matricellular proteins that provide bioinductive effects on local cells that influence phenotype and behaviors. Bioactive acellular ECM scaffolds can be used therapeutically to stimulate adaptive tissue repair. Fibroblast growth factor-2 (FGF-2) attenuates transforming growth factor-ß1 (TGF-ß1)-mediated cardiac fibrosis. Heparin glycosaminoglycan can influence FGF-2 bioactivity and could be leveraged to enhance tissue engineering strategies. We explored the effects of heparin on FGF-2 enhancement of bioactive ECM scaffold biomaterials for its antifibrotic effect on attenuating human cardiac myofibroblast activation. Increasing heparin concentration at a fixed concentration of FGF-2 markedly increased the amount of FGF-2 retained and eluted by ECM scaffolds. To explore synergistic bioinductive effects of heparin and FGF-2, collagen gel contraction assay using human cardiac myofibroblasts was performed in vitro. Myofibroblast activation was induced by profibrotic cytokine, TGF-ß1. FGF-2 and heparin in combination reduced human cardiac myofibroblast-mediated collagen gel contraction to a greater extent than FGF-2 alone. These observations were confirmed for both human atrial and human ventricular cardiac fibroblasts. Cell death was not different between groups. In summary, heparin is an effective adjuvant to enhance FGF-2 loading and elution of acellular ECM scaffold biomaterials. Heparin increases the bioactive effects of FGF-2 in attenuating human cardiac myofibroblast activation in response to profibrotic TGF-ß1. These data may inform tissue engineering strategies for myocardial repair to prevent fibrosis.

Bioactive Extracellular Matrix Scaffold Promotes Adaptive Cardiac Remodeling and Repair.

Structural cardiac remodeling after ischemic injury can induce a transition to heart failure from progressive loss of cardiac function. Cellular regenerative therapies are promising but face significant translational hurdles. Tissue extracellular matrix (ECM) holds the necessary environmental cues to stimulate cell-based endogenous myocardial repair pathways and promote adaptive remodeling toward functional recovery. Heart epicardium has emerged as an important anatomic niche for endogenous repair pathways including vasculogenesis and cardiogenesis. We show that acellular ECM scaffolds surgically implanted on the epicardium following myocardial infarction (MI) can attenuate structural cardiac remodeling and improve functional recovery. We assessed the efficacy of this strategy on post-MI functional recovery by comparing intact bioactive scaffolds with biologically inactivated ECM scaffolds. We confirm that bioactive properties within the acellular ECM biomaterial are essential for the observed functional benefits. We show that interaction of human cardiac fibroblasts with bioactive ECM can induce a robust cell-mediated vasculogenic paracrine response capable of functional blood vessel assembly. Fibroblast growth factor-2 is uncovered as a critical regulator of this novel bioinductive effect. Acellular bioactive ECM scaffolds surgically implanted on the epicardium post-MI can reprogram resident fibroblasts and stimulate adaptive pro-reparative pathways enhancing functional recovery. We introduce a novel surgical strategy for tissue repair that can be performed as an adjunct to conventional surgical revascularization with minimal translational challenges.

Role of the pH in state-dependent blockade of hERG currents.

Mutations that reduce inactivation of the voltage-gated Kv11.1 potassium channel (hERG) reduce binding for a number of blockers. State specific block of the inactivated state of hERG block may increase risks of drug-induced Torsade de pointes. In this study, molecular simulations of dofetilide binding to the previously developed and experimentally validated models of the hERG channel in open and open-inactivated states were combined with voltage-clamp experiments to unravel the mechanism(s) of state-dependent blockade. The computations of the free energy profiles associated with the drug block to its binding pocket in the intra-cavitary site display startling differences in the open and open-inactivated states of the channel. It was also found that drug ionization may play a crucial role in preferential targeting to the open-inactivated state of the pore domain. pH-dependent hERG blockade by dofetilie was studied with patch-clamp recordings. The results show that low pH increases the extent and speed of drug-induced block. Both experimental and computational findings indicate that binding to the open-inactivated state is of key importance to our understanding of the dofetilide's mode of action.

Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-ß1.

Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-ß1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-ß blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-ß-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-ß1. These data implicate inflammation as a potential driver of cardiac fibrosis.

Fibroblast growth factor-2 regulates human cardiac myofibroblast-mediated extracellular matrix remodeling.

BACKGROUND: Tissue fibrosis and chamber remodeling is a hallmark of the failing heart and the final common pathway for heart failure of diverse etiologies. Sustained elevation of pro-fibrotic cytokine transforming growth factor-beta1 (TGFß1) induces cardiac myofibroblast-mediated fibrosis and progressive structural tissue remodeling. OBJECTIVES: We examined the effects of low molecular weight fibroblast growth factor (LMW-FGF-2) on human cardiac myofibroblast-mediated extracellular matrix (ECM) dysregulation and remodeling. METHODS: Human cardiac biopsies were obtained during open-heart surgery and myofibroblasts were isolated, passaged, and seeded within type I collagen matrices. To induce myofibroblast activation and ECM remodeling, myofibroblast-seeded collagen gels were exposed to TGFß1. The extent of ECM contraction, myofibroblast activation, ECM dysregulation, and cell apoptosis was determined in the presence of LMW-FGF-2 and compared to its absence. Using a novel floating nylon-grid supported thin collagen gel culture platform system, myofibroblast activation and local ECM remodeling around isolated single cells was imaged using confocal microscopy and quantified by image analysis. RESULTS: TGFß1 induced significant myofibroblast activation and ECM dysregulation as evidenced by collagen gel contraction, structural ECM remodeling, collagen synthesis, ECM degradation, and altered TIMP expression. LMW-FGF-2 significantly attenuated TGFß1 induced myofibroblast-mediated ECM remodeling. These observations were similar using either ventricular or atrial-derived cardiac myofibroblasts. In addition, for the first time using individual cells, LMW-FGF-2 was observed to attenuate cardiac myofibroblast activation and prevent local cell-mediated ECM perturbations. CONCLUSIONS: LMW-FGF-2 attenuates human cardiac myofibroblast-mediated ECM remodeling and may prevent progressive maladaptive chamber remodeling and tissue fibrosis for patients with diverse structural heart diseases.

Tetrandrine reverses human cardiac myofibroblast activation and myocardial fibrosis.

Tetrandrine (TTD) is a calcium channel blocker with documented antifibrotic actions. In this study, for the first time, we identified that TTD can directly prevent in vitro human cardiac myofibroblast activation and limit in vivo myocardial fibrosis. In vitro, cardiac myofibroblasts from human atrial biopsies (N = 10) were seeded in three-dimensional collagen matrices. Cell-collagen constructs were exposed to transforming growth factor-ß1 (10 ng/ml), with or without TTD (1 and 5 µM) for 48 h. Collagen gel contraction, myofibroblast activation (α-smooth muscle actin expression), expression of profibrotic mRNAs, and rate of collagen protein synthesis were compared. TTD decreased collagen gel contraction (79.7 ± 1.3 vs 60.1 ± 8.9%, P < 0.01), α-smooth muscle actin expression (flow cytometry), collagen synthesis ([(3)H]proline incorporation), and collagen mRNA expression. Cell viability was similar between groups (annexin positive cells: 1.7 vs. 1.4%). TTD inhibited collagen gel contraction in the presence of T-type and L-type calcium channel blockers, and the intracellular calcium chelator BAPTA-AM (15 µM), suggesting that the observed effects are not mediated by calcium homeostasis. In vivo, Dahl salt-sensitive hypertensive rats were treated with variable doses of TTD (by intraperitoneal injection over 4 wk) and compared with untreated controls (N = 12). Systemic blood pressure was monitored by tail cuff. Myocardial fibrosis and left ventricular compliance were assessed by histology and passive pressure-volume analysis. Myocardial fibrosis was attenuated compared with untreated controls (%collagen area: 9.4 ± 7.3 vs 2.1 ± 1.0%, P < 0.01). Left ventricular compliance was preserved. In conclusion, TTD reverses human cardiac myofibroblast activation and myocardial fibrosis, independent of calcium channel blockade.

Role of mutation and pharmacologic block of human KCNH2 in vasculogenesis and fetal mortality: partial rescue by transforming growth factor-ß.

BACKGROUND: N629D KCNH2 is a human missense long-QT2 mutation. Previously, we reported that the N629D/N629D mutation embryos disrupted cardiac looping, right ventricle development, and ablated IKr activity at E9.5. The present study evaluates the role of KCNH2 in vasculogenesis. METHODS AND RESULTS: N629D/N629D yolk sac vessels and aorta consist of sinusoids without normal arborization. Isolated E9.5 +/+ first branchial arches showed normal outgrowth of mouse ERG-positive/α-smooth muscle actin coimmunolocalized cells; however, outgrowth was grossly reduced in N629D/N629D. N629D/N629D aortas showed fewer α-smooth muscle actin positive cells that were not coimmunolocalized with mouse ERG cells. Transforming growth factor-ß treatment of isolated N629D/N629D embryoid bodies partially rescued this phenotype. Cultured N629D/N629D embryos recapitulate the same cardiovascular phenotypes as seen in vivo. Transforming growth factor-ß treatment significantly rescued these embryonic phenotypes. Both in vivo and in vitro, dofetilide treatment, over a narrow window of time, entirely recapitulated the N629D/N629D fetal phenotypes. Exogenous transforming growth factor-ß treatment also rescued the dofetilide-induced phenotype toward normal. CONCLUSIONS: Loss of function of KCNH2 mutations results in defects in cardiogenesis and vasculogenesis. Because many medications inadvertently block the KCNH2 potassium current, these novel findings seem to have clinical relevance.

Human cardiac fibroblast extracellular matrix remodeling: dual effects of tissue inhibitor of metalloproteinase-2.

OBJECTIVE: Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinases (MMPs) that attenuates maladaptive cardiac remodeling in ischemic heart failure. We examined the effects of TIMP-2 on human cardiac fibroblast activation and extracellular matrix (ECM) remodeling. METHODS: Human cardiac fibroblasts within a three-dimensional collagen matrix were assessed for phenotype conversion, ECM architecture and key molecular regulators of ECM remodeling after differential exposure to TIMP-2 and Ala+TIMP-2 (a modified TIMP-2 analogue devoid of MMP inhibitory activity). RESULTS: TIMP-2 induced opposite effects on human cardiac fibroblast activation and ECM remodeling depending on concentration. TIMP-2 activated fibroblasts into contractile myofibroblasts that remodeled ECM. At higher concentrations (>10 nM), TIMP-2 inhibited fibroblast activation and prevented ECM remodeling. As compared to profibrotic cytokine transforming growth factor (TGF)-beta1, TIMP-2 activated fibroblasts and remodeled ECM without a net accumulation of matrix elements. TIMP-2 increased total protease activity as compared to TGF-beta1. Ala+TIMP-2 exposure revealed that the actions of TIMP-2 on cardiac fibroblast activation are independent of its effects on MMP inhibition. In the presence of GM6001, a broad-spectrum MMP inhibitor, TIMP-2-mediated ECM contraction was completely abolished, indicating that TIMP-2-mediated fibroblast activation is MMP dependent. CONCLUSION: TIMP-2 functions in a contextual fashion such that the effect on cardiac fibroblasts depends on the tissue microenvironment. These observations highlight potential clinical challenges in using TIMP-2 as a therapeutic strategy to attenuate postinjury cardiac remodeling.

Exaggerated block of hERG (KCNH2) and prolongation of action potential duration by erythromycin at temperatures between 37 degrees C and 42 degrees C.

BACKGROUND: Environmental and genetic factors interact to define susceptibility to drug-induced long QT syndrome. Although erythromycin induces long QT syndrome, substantial variability exists with regard to its incidence. OBJECTIVES: Because fever frequently results in empiric antibiotic usage, we assessed whether temperatures over the range from 36 degrees to 42 degrees C determined responsiveness to erythromycin (100 micromol/L). METHODS: I(hERG) was recorded in mammalian cells, and action potentials were recorded in neonatal ventricular mouse myocytes. RESULTS: Erythromycin (100 micromol/L) produced no block of I(hERG) at 22 degrees C but produced significant block at 37 degrees C. Extent of block of I(hERG) increased linearly (r = 0.46, P < .01) as temperature increased between 36 degrees C and 42 degrees C. To assess physiologic relevance, action potential duration (APD) was recorded at temperatures between 36 degrees C and 42 degrees C in neonatal ventricular myocytes. Significantly greater prolongation of APD by erythromycin was observed at 42 degrees C compared with 37 degrees C. To assess whether transmembrane diffusion of erythromycin was the rate-limiting step for block of I(hERG) at 22 degrees C, erythromycin was applied within the patch pipette. Under these conditions, erythromycin rapidly blocked I(hERG) even at 22 degrees C. The F656C mutation in the distal S6 of KCNH2 completely abrogated block of I(hERG) measured at 37 degrees C. CONCLUSION: Progressively greater block of hERG and prolongation of APD by erythromycin was observed at temperatures between 36 and 42 degrees C. Temperature-dependent block of I(hERG) is explained by temperature-dependent access of erythromycin to the intracellular binding site at F656.

Prolonged repolarization and triggered activity induced by adenoviral expression of HERG N629D in cardiomyocytes derived from stem cells.

OBJECTIVE: The long QT syndrome, N629D HERG mutation, alters the pore selectivity signature sequence, GFGN to GFGD. Heterologous co-expression of N629D and the wildtype HERG resulted in a relative loss of the selectivity of K+ over Na+, but its physiologic relevance has not been assessed in cardiac myocytes. METHODS AND RESULTS: Accordingly, N629D was overexpressed, via adenoviral gene transfer, in cardiomyocytes derived from mouse stem cells. Three IKr phenotypes were observed: (1) the wildtype-like IKr showed inward rectification and a positive tail current; (2) the N629D-like IKr showed outward rectification and an inward tail current; and (3) intermediate IKr showed a small outward tail current. Action potentials (AP) were paired with the IKr measurements in each cell. Resting membrane potential (RMP) was critically dependent on the IKr phenotype. The resting membrane potential of the cells was -61 +/- 5 mV (n=40) in wildtype, -63 +/- 3 mV (n=18) in wildtype-like IKr phenotype, -30 +/- 2 mV (n=12) in N629D-like and -47 +/- 2 mV (n=24) in intermediate phenotype (p<0.00001). Triggered action potential durations (APD) were: 62 +/- 12 ms (n=6) in wildtype, 65 +/- 11 ms (n=6) in wildtype-like IKr phenotypes and 106 +/- 10 ms (n=6) (p<0.01) in intermediate IKr phenotypes. Lowering [K+]o hyperpolarized wildtype cells and cells with a wildtype-like IKr phenotype, but depolarized those with intermediate phenotype (from -45 +/- 1 to -35 +/- 0.5 mV (n=12), p<0.01). In 6 of 12 cells, with intermediate phenotype, the hypokalemia-induced depolarization resulted in triggered activity. TTX suppressed this triggered activity. CONCLUSION: Overexpression of N629D in cardiomyocytes derived from stem cells results in phenotypic variability in IKr, which was the critical determinant of the resting membrane potential, action potential duration and arrhythmogenic response to low [K+]o.