Smoothelin-like 1 knockout mice display sex-dependent alterations in blood flow and cardiac function.

Smoothelin-like 1 (SMTNL1) modulates the contractile performance of smooth muscle and thus has a key role in vascular homeostasis. Elevated vascular tone, recognized as a contributor to the development of progressive cardiac dysfunction, was previously found with SMTNL1 deletion. In this study, we assessed cardiac morphology and function of male and female, wild-type (Smtnl1+/+) and global SMTNL1 knockout (Smtnl1-/-) mice at 10 weeks of age. Gross dissection revealed distinct cardiac morphology only in males; Smtnl1-/- hearts were significantly smaller than Smtnl1+/+, but the left ventricle (LV) proportion of heart mass was greater. Male Smtnl1-/- mice also displayed increased ejection fraction and fractional shortening, as well as elevated aortic and pulmonary flow velocities. The impact of cardiac stress with pressure overload by transverse aortic constriction (TAC) was examined in male mice. With TAC banding, systolic function was preserved, but the LV filling pressure was selectively elevated due to relaxation impairment. Smtnl1-/- mice displayed higher early/passive filling velocity of LV/early mitral annulus velocity ratio (E/E' ratio) and myocardial performance index along with a prolonged isovolumetric relaxation time. Taken together, the findings support a novel, sex-dimorphic role for SMTNL1 in modulating cardiac structure and function of mice.

Loss of insulin signaling may contribute to atrial fibrillation and atrial electrical remodeling in type 1 diabetes.

Atrial fibrillation (AF) is prevalent in diabetes mellitus (DM); however, the basis for this is unknown. This study investigated AF susceptibility and atrial electrophysiology in type 1 diabetic Akita mice using in vivo intracardiac electrophysiology, high-resolution optical mapping in atrial preparations, and patch clamping in isolated atrial myocytes. qPCR and western blotting were used to assess ion channel expression. Akita mice were highly susceptible to AF in association with increased P-wave duration and slowed atrial conduction velocity. In a second model of type 1 DM, mice treated with streptozotocin (STZ) showed a similar increase in susceptibility to AF. Chronic insulin treatment reduced susceptibility and duration of AF and shortened P-wave duration in Akita mice. Atrial action potential (AP) morphology was altered in Akita mice due to a reduction in upstroke velocity and increases in AP duration. In Akita mice, atrial Na+ current (INa) and repolarizing K+ current (IK) carried by voltage gated K+ (Kv1.5) channels were reduced. The reduction in INa occurred in association with reduced expression of SCN5a and voltage gated Na+ (NaV1.5) channels as well as a shift in INa activation kinetics. Insulin potently and selectively increased INa in Akita mice without affecting IK Chronic insulin treatment increased INa in association with increased expression of NaV1.5. Acute insulin also increased INa, although to a smaller extent, due to enhanced insulin signaling via phosphatidylinositol 3,4,5-triphosphate (PIP3). Our study reveals a critical, selective role for insulin in regulating atrial INa, which impacts susceptibility to AF in type 1 DM.

Distinct patterns of atrial electrical and structural remodeling in angiotensin II mediated atrial fibrillation.

Atrial fibrillation (AF) is prevalent in hypertension and elevated angiotensin II (Ang II); however, the mechanisms by which Ang II leads to AF are poorly understood. Here, we investigated the basis for this in mice treated with Ang II or saline for 3 weeks. Ang II treatment increased susceptibility to AF compared to saline controls in association with increases in P wave duration and atrial effective refractory period, as well as reductions in right and left atrial conduction velocity. Patch-clamp studies demonstrate that action potential (AP) duration was prolonged in right atrial myocytes from Ang II treated mice in association with a reduction in repolarizing K+ currents. In contrast, APs in left atrial myocytes from Ang II treated mice showed reductions in upstroke velocity and overshoot, as well as greater prolongations in AP duration. Ang II reduced Na+ current (INa) in the left, but not the right atrium. This reduction in INa was reversible following inhibition of protein kinase C (PKC) and PKCα expression was increased selectively in the left atrium in Ang II treated mice. The transient outward K+ current (Ito) showed larger reductions in the left atrium in association with a shift in the voltage dependence of activation. Finally, Ang II caused fibrosis throughout the atria in association with changes in collagen expression and regulators of the extracellular matrix. This study demonstrates that hypertension and elevated Ang II cause distinct patterns of electrical and structural remodeling in the right and left atria that collectively create a substrate for AF.

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.

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.

Enhanced cardiac protein glycosylation (O-GlcNAc) of selected mitochondrial proteins in rats artificially selected for low running capacity.

O-linked ß-N-acetyl glucosamine (O-GlcNAc) is a posttranslational modification consisting of a single N-acetylglucosamine moiety attached by an O-ß-glycosidic linkage to serine and threonine residues of both nuclear and cytosolic proteins. Analogous to phosphorylation, the modification is reversible and dynamic, changing in response to stress, nutrients, hormones, and exercise. Aims of this study were to examine differences in O-GlcNAc protein modification in the cardiac tissue of rats artificially selected for low (LCR) or high (HCR) running capacity. Hyperinsulinemic-euglycemic clamps in conscious animals assessed insulin sensitivity while 2-[(14)C] deoxyglucose tracked both whole body and tissue-specific glucose disposal. Immunoblots of cardiac muscle examined global O-GlcNAc modification, enzymes that control its regulation (OGT, OGA), and specific proteins involved in mitochondrial oxidative phosphorylation. LCR rats were insulin resistant disposing of 65% less glucose than HCR. Global tissue O-GlcNAc, OGT, OGA, and citrate synthase were similar between groups. Analysis of cardiac proteins revealed enhanced O-GlcNAcylation of mitochondrial Complex I, Complex IV, VDAC, and SERCA in LCR compared with HCR. These results are the first to establish an increase in specific protein O-GlcNAcylation in LCR animals that may contribute to progressive mitochondrial dysfunction and the pathogenesis of insulin resistance observed in the LCR phenotype.

PDE4D mediates impaired ß-adrenergic receptor signalling in the sinoatrial node in mice with hypertensive heart disease.

AIMS: The sympathetic nervous system increases HR by activating ß-adrenergic receptors (ß-ARs) and increasing cAMP in sinoatrial node (SAN) myocytes while phosphodiesterases (PDEs) degrade cAMP. Chronotropic incompetence, the inability to regulate heart rate (HR) in response to sympathetic nervous system activation, is common in hypertensive heart disease; however, the basis for this is poorly understood. The objective of this study was to determine the mechanisms leading to chronotropic incompetence in mice with angiotensin II (AngII)-induced hypertensive heart disease. METHODS AND RESULTS: C57BL/6 mice were infused with saline or AngII (2.5 mg/kg/day for 3 weeks) to induce hypertensive heart disease. HR and SAN function in response to the ß-AR agonist isoproterenol (ISO) were studied in vivo using telemetry and electrocardiography, in isolated atrial preparations using optical mapping, in isolated SAN myocytes using patch-clamping, and using molecular biology. AngII-infused mice had smaller increases in HR in response to physical activity and during acute ISO injection. Optical mapping of the SAN in AngII-infused mice demonstrated impaired increases in conduction velocity and altered conduction patterns in response to ISO. Spontaneous AP firing responses to ISO in isolated SAN myocytes from AngII-infused mice were impaired due to smaller increases in diastolic depolarization (DD) slope, hyperpolarization-activated current (If), and L-type Ca2+ current (ICa,L). These changes were due to increased localization of PDE4D surrounding ß1- and ß2-ARs in the SAN, increased SAN PDE4 activity, and reduced cAMP generation in response to ISO. Knockdown of PDE4D using a virus-delivered shRNA or inhibition of PDE4 with rolipram normalized SAN sensitivity to ß-AR stimulation in AngII-infused mice. CONCLUSIONS: AngII-induced hypertensive heart disease results in impaired HR responses to ß-AR stimulation due to up-regulation of PDE4D and reduced effects of cAMP on spontaneous AP firing in SAN myocytes.

Glucagon-Like Peptide-1 Protects Against Atrial Fibrillation and Atrial Remodeling in Type 2 Diabetic Mice.

Atrial fibrillation (AF) is highly prevalent in type 2 diabetes where it increases morbidity and mortality. Glucagon-like peptide (GLP)-1 receptor agonists are used in the treatment of type 2 diabetes (T2DM), but their effects on AF in T2DM are poorly understood. The present study demonstrates type 2 diabetic db/db mice are highly susceptible to AF in association with atrial electrical and structural remodeling. GLP-1, as well as the long-acting GLP-1 analogue liraglutide, reduced AF and prevented atrial remodeling in db/db mice. These data suggest that GLP-1 and related analogues could protect against AF in patients with T2DM.

Natriuretic Peptide Receptor B Protects Against Atrial Fibrillation by Controlling Atrial cAMP Via Phosphodiesterase 2.

BACKGROUND: ß-AR (ß-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca2+ homeostasis via cAMP-dependent mechanisms; however, enhanced ß-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca2+ homeostasis, and atrial arrhythmogenesis is incompletely understood. METHODS: Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B+/-) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca2+ imaging, and in mouse and human atrial tissues using molecular biology. RESULTS: Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B+/- mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B+/- mice leading to larger increases in atrial cAMP in the presence of the ß-AR agonist isoproterenol. NPR-B+/- mice displayed larger increases in action potential duration and L-type Ca2+ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca2+ release events and delayed afterdepolarizations in NPR-B+/- atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B+/- atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca2+ current through PDE2 in mouse and human atrial myocytes. CONCLUSIONS: NPR-B protects against AF by preventing enhanced atrial responses to ß-adrenergic receptor agonists.

Loss of Natriuretic Peptide Receptor C Enhances Sinoatrial Node Dysfunction in Aging and Frail Mice.

Heart rate (HR) is controlled by the sinoatrial node (SAN). SAN dysfunction is highly prevalent in aging; however, not all individuals age at the same rate. Rather, health status during aging is affected by frailty. Natriuretic peptides regulate SAN function in part by activating natriuretic peptide receptor C (NPR-C). The impacts of NPR-C on HR and SAN function in aging and as a function of frailty are unknown. Frailty was measured in aging wild-type and NPR-C knockout (NPR-C-/-) mice using a mouse clinical frailty index (FI). HR and SAN structure and function were investigated using intracardiac electrophysiology in anesthetized mice, high-resolution optical mapping in intact atrial preparations, histology, and molecular biology. NPR-C-/- mice rapidly became frail leading to shortened life span. HR was reduced and SAN recovery time was increased in older versus younger mice, and these changes were exacerbated in NPR-C-/- mice; however, there was substantial variability among age groups and genotypes. HR and SAN recovery time were correlated with FI score and fell along a continuum regardless of age or genotype. Optical mapping demonstrates impairments in SAN function that were also correlated with FI score. SAN fibrosis was increased in aged and NPR-C-/- mice and was graded by FI score. Loss of NPR-C results in accelerated aging and rapid decline in health status in association with impairments in HR and SAN function. Frailty assessment was effective and better able to distinguish aging-dependent changes in SAN function in the setting of shortened life span due to loss of NPR-C.

The T-type Calcium Channel Cav3.1 in Y79 Retinoblastoma Cells is Regulated by the Epidermal Growth Factor Receptor via the MAPK Signaling Pathway.

PURPOSE: Retinoblastoma is the most frequent intraocular cancer in children. It is also one of the most common causes for enucleation and carries a significant morbidity rate in affected individuals. Hence, studies on its pathophysiological and growth regulatory mechanisms are urgently needed to identify more effective novel therapeutics. METHODS: Using the Y79 retinoblastoma cell line, we investigated the electrophysiological and functional activities of the T-type voltage-gated calcium channel Cav3.1, that is constitutively expressed in these cells. We also analyzed the Akt and MAPK signaling pathways downstream of the epidermal growth factor receptor (EGFR) to understand the mechanism responsible for the inhibition of Cav3.1. RESULTS: We demonstrate that the EGFR inhibitor Afatinib significantly reduced cell viability and Cav3.1 mRNA expression and electrophysiological activity. At low concentrations (1 µM), Afatinib reduced the amplitude of Cav3.1 current density, whereas at a high concentration (10 µM), it completely abolished the voltage-gated calcium current. Our results show that inhibition of the MAPK pathway by a specific inhibitor VX-11e affected the Cav3.1 current in a dose-dependent manner. VX-11e (50 nM-1 µM) treatment reduced Cav3.1 current densities in Y79 cells, with complete abolishment of Cav3.1 current at higher concentrations (5 µM). We also demonstrate that the specific inhibition of the Akt kinase (using MK-2206) had no effect on the Cav3.1 currents. CONCLUSION: Our study provides a functional relationship between the MAPK pathway and EGFR signaling and indicates that the MAPK signaling pathway mediates the control of Cav3.1 by EGFR in retinoblastoma.

Natriuretic peptide receptor B maintains heart rate and sinoatrial node function via cyclic GMP-mediated signalling.

AIMS: Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP), have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signalling in regulating HR and SAN function. METHODS AND RESULTS: We have used NPR-B deficient mice (NPR-B+/-) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anaesthetized mice, high-resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/- mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/- mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signalling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/- mice. NPR-B+/- mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function. CONCLUSIONS: NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signalling mechanism.

Two-pore domain K⁺ channels regulate membrane potential of isolated human articular chondrocytes.

Potassium channels that regulate resting membrane potential (RMP) of human articular chondrocytes (HACs) of the tibial joint maintained in short-term (0-3 days) non-confluent cell culture were studied using patch-clamp techniques. Quantitative PCR showed that transcripts of genes for two-pore domain K(+) channels (KCNK1, KCNK5 and KCNK6), and 'BK' Ca(2+)-activated K(+) channels (KCNMA1) were abundantly expressed. Immunocytological methods detected α-subunits for BK and K(2p)5.1 (TASK-2) K(+) channels. Electrophysiological recordings identified three distinct K(+) currents in isolated HACs: (i) a voltage- and time-dependent 'delayed rectifier', blocked by 100 nM α-dendrotoxin, (ii) a large 'noisy' voltage-dependent current that was blocked by low concentrations of tetraethylammonium (TEA; 50% blocking dose = 0.15 mM) and iberiotoxin (52% block, 100 nM) and (iii) a voltage-independent 'background' K(+) current that was blocked by acidic pH (5.5-6), was increased by alkaline pH (8.5), and was not blocked by TEA, but was blocked by the local anaesthetic bupivacaine (0.25 mM). The RMP of isolated HACs was very slightly affected by 5 mM TEA, which was sufficient to block both voltage-dependent K(+) currents, suggesting that these currents probably contributed little to maintaining RMP under 'resting' conditions (i.e. low internal [Ca(2+)]). Increases in external K(+) concentration depolarized HACs by 30 mV in response to a 10-fold increase in [K(+)], indicating a significant but not exclusive role for K(+) current in determining RMP. Increases in external [K(+)] in voltage-clamped HACs revealed a voltage-independent K(+) current whose inward current magnitude increased with external [K(+)]. Block of this current by bupivacaine (0.25-1 mM) in 5 and 25 mM external [K(+)] resulted in a large (8-25 mV) depolarization of RMP. The biophysical and pharmacological properties of the background K(+) current, together with expression of mRNA and α-subunit protein for TASK-2, strongly suggest that these two-pore domain K(+) channels contribute significantly to stabilizing the RMP of HACs.

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.

Electrical and structural remodeling contribute to atrial fibrillation in type 2 diabetic db/db mice.

BACKGROUND: Atrial fibrillation (AF) is highly prevalent in diabetes mellitus (DM), yet the basis for this finding is poorly understood. Type 2 DM may be associated with unique patterns of atrial electrical and structural remodeling; however, this has not been investigated in detail. OBJECTIVE: The purpose of this study was to investigate AF susceptibility and atrial electrical and structural remodeling in type 2 diabetic db/db mice. METHODS: AF susceptibility and atrial function were assessed in male and female db/db mice and age-matched wildtype littermates. Electrophysiological studies were conducted in vivo using intracardiac electrophysiology and programmed stimulation. Atrial electrophysiology was also investigated in isolated atrial preparations using high-resolution optical mapping and in isolated atrial myocytes using patch-clamping. Molecular biology studies were performed using quantitative polymerase chain reaction and western blotting. Atrial fibrosis was assessed using histology. RESULTS: db/db mice were highly susceptible to AF in association with reduced atrial conduction velocity, action potential duration prolongation, and increased heterogeneity in repolarization in left and right atria. In db/db mice, atrial K+ currents, including the transient outward current (Ito) and the ultrarapid delayed rectifier current (IKur), were reduced. The reduction in Ito occurred in association with reductions in Kcnd2 mRNA expression and KV4.2 protein levels. The reduction in IKur was not related to gene or protein expression changes. Interstitial atrial fibrosis was increased in db/db mice. CONCLUSION: Our study demonstrates that increased susceptibility to AF in db/db mice occurs in association with impaired electrical conduction as well as electrical and structural remodeling of the atria.

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.

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.

Adeno-associated virus-mediated expression of thyroid hormone receptor isoforms-alpha1 and -beta1 improves contractile function in pressure overload-induced cardiac hypertrophy.

Pressure overload-induced cardiac hypertrophy leads to decreased contractile performance, frequently progressing to heart failure. Cardiac hypertrophy and heart failure can be accompanied by the so-called sick thyroid syndrome, resulting in decreased serum T(3) levels along with decreased expression of thyroid hormone receptors (TRalpha1 and TRbeta1) and sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Because the binding of T(3) occupied receptors to the thyroid response elements in the SERCA promotor can increase gene expression, we wanted to determine whether increasing TR expression in the hypertrophied heart could also improve SERCA expression and cardiac function. Mice subjected to aortic constriction to generate pressure overload-induced hypertrophy were also subjected to gene therapy using adeno-associated virus (AAV) expressing either TRalpha1 or TRbeta1, with LacZ expressing AAV serving as control. After 8 wk of aortic constriction, a similar degree of hypertrophy was observed in all three groups; however, mice treated with TRalpha1 or TRbeta1 showed improved contractile function. Administration of a physiological dose of T(3) increased serum T(3) levels only into the lower range of normal. This T(3) dose, with or without AAV TR treatment, did not result in any significant increase in contractile performance. Calcium transients measured in isolated myocytes also exhibited an enhanced rate of decay associated with TRalpha1 or TRbeta1 treatment. Western blot analysis showed increased SERCA expression in the TRalpha1- or TRbeta1-treated groups relative to the LacZ-treated control group. These results demonstrate that increasing TR expression in the hypertrophied heart is associated with an improvement in contractile function and increased SERCA expression.