Dedifferentiation of Human Cardiac Myofibroblasts Is Independent of Activation of COX-2/PGE2 Pathway.

The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in activation and progression of cardiac fibrosis in heart disease. TGF-ß is one of the key cytokines that promotes transition of fibroblasts to myofibroblasts. Dedifferentiation of formed myofibroblasts or reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Prostaglandin E2 (PGE2) has been shown to dedifferentiate human lung myofibroblasts. The role of activation of the COX-2/PGE2 pathway in dedifferentiation of cardiac myofibroblasts remains unknown. Here, we show that phorbol 12-myristate 13-acetate (PMA) but not PGE2 induces dedifferentiation of de novo adult human cardiac myofibroblasts stimulated by TGF-ß1 from human cardiac fibroblasts as evidenced by reduced expression of α-smooth muscle actin (α-SMA). PMA remarkably increased endogenous levels of PGE2 in human cardiac myofibroblasts. Pretreatment of myofibroblasts with NS-398, a selective COX-2 inhibitor, and PF-04418948, a selective PGE2 receptor type 2 (EP2) antagonist, had no effect on expression of α-SMA nor abolished the dedifferentiation induced by PMA. Our results indicated that endogenous and exogenous PGE2 has no effects on dedifferentiation of cardiac myofibroblasts. PMA-induced dedifferentiation of cardiac myofibroblast is independent of activation of COX-2 and PGE2 pathway. The mechanism in PMA-induced reversal of cardiac myofibroblasts needs to be explored further.

MicroRNA targets and biomarker validation for diabetes-associated cardiac fibrosis.

Cardiac fibrosis is one of the main characteristics of diabetic cardiomyopathy and manifests excessive accumulation of extracellular matrix proteins in the heart. Several signaling pathways have been proposed for pathogenesis of cardiac fibrosis in the diabetic heart. TGF-ß/Smad2/3-dependent or independent pathway is the major signaling molecule core in the pathogenesis of cardiac fibrosis. MicroRNAs (miRNAs, miR) are ~22-nuceotide regulatory RNAs that are involved in gene silencing through the degradation of post-transcriptional mRNA or suppression of the expressed proteins. Hyperglycemia in the diabetic heart regulates expression of some miRNAs. Target molecules of miRNAs can be identified through biocomputational database initial screening and dual luciferase assay validation. miR-21, miR-150-5p, miR-155, miR-216a-3p, miR-221-3p, miR-223, and miR-451 were up-regulated in the diabetic heart and promoted cardiac fibrosis through targeting signaling pathways in cardiac fibroblasts, endothelial cells, and cardiac myocytes. miR-15a/-15b, miR-18a-5p, miR-20a-5p, miR-26b-5p, miR-29, miR-133a, miR-141, miR-146, miR-200b, miR-203, miR-222, and miR-551b-5p were down-regulated in the diabetic heart and exhibited anti-fibrosis when they were overexpressed. miRNAs are stable molecules and may reflect the pathological changes of organs. Some miRNAs have been detected in the plasma or serum in patients with diabetes mellitus or heart failure. Exploration of targets and biomarkers of miRNA may provide additional information on pathogenesis and diagnosis of cardiac fibrosis and novel targets to tackle diabetic cardiomyopathy.

Targeted deletion of T-cell S1P receptor 1 ameliorates cardiac fibrosis in streptozotocin-induced diabetic mice.

Infiltration of T cells is associated with patients who have diabetes at an increased risk of heart attack. T-cell sphingosine 1-phosphate receptor 1 (S1P1)-mediated signaling directs T-lymphocyte trafficking. Effects of T-cell S1P1 activation on cardiac fibrosis in a murine diabetic model remain to be explored. For this purpose, conditional T-cell S1P1 knockout (TS1P1KO) mice generated by crossing S1pr1loxP/loxP mice with Lck-Cre mice were used in a model of streptozotocin-induced diabetic cardiomyopathy. The TS1P1KO mice exhibited sustained deficiency of both CD4+ and CD8+ T cells in the blood. The TS1P1KO vehicle control mouse hearts featured an altered phenotype characterized by increased myocardial fibrosis and reduced cardiac contractility under normal levels of glucose. Compared with littermate diabetic mice, TS1P1KO diabetic mice had improved cardiac function and alleviated cardiac fibrosis detected after 11 wk of diabetic induction. Our results indicate that T-cell S1P1 signaling activation plays a dual role in the pathogenesis of cardiac fibrosis with respect to the levels of glucose: T-cell S1P1 activation exerts antifibrotic effects in normoglycemia but exacerbates fibrosis under hyperglycemia.-Abdullah, C. S., Jin, Z.-Q. Targeted deletion of T-cell S1P receptor 1 ameliorates cardiac fibrosis in streptozotocin-induced diabetic mice.

Ischemic preconditioning enhances integrity of coronary endothelial tight junctions.

Ischemic preconditioning (IPC) is one of the most effective procedures known to protect hearts against ischemia/reperfusion (IR) injury. Tight junction (TJ) barriers occur between coronary endothelial cells. TJs provide barrier function to maintain the homeostasis of the inner environment of tissues. However, the effect of IPC on the structure and function of cardiac TJs remains unknown. We tested the hypothesis that myocardial IR injury ruptures the structure of TJs and impairs endothelial permeability whereas IPC preserves the structural and functional integrity of TJs in the blood-heart barrier. Langendorff hearts from C57BL/6J mice were prepared and perfused with Krebs-Henseleit buffer. Cardiac function, creatine kinase release, and myocardial edema were measured. Cardiac TJ function was evaluated by measuring Evans blue-conjugated albumin (EBA) content in the extravascular compartment of hearts. Expression and translocation of zonula occludens (ZO)-2 in IR and IPC hearts were detected with Western blot. A subset of hearts was processed for the observation of ultra-structure of cardiac TJs with transmission electron microscopy. There were clear TJs between coronary endothelial cells of mouse hearts. IR caused the collapse of TJs whereas IPC sustained the structure of TJs. IR increased extravascular EBA content in the heart and myocardial edema but decreased the expression of ZO-2 in the cytoskeleton. IPC maintained the structure of TJs. Cardiac EBA content and edema were reduced in IPC hearts. IPC enhanced the translocation of ZO-2 from cytosol to cytoskeleton. In conclusion, TJs occur in normal mouse heart. IPC preserves the integrity of TJ structure and function that are vulnerable to IR injury.

S1P lyase: a novel therapeutic target for ischemia-reperfusion injury of the heart.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia (n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls (n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity (n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts (n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P1 and S1P3 receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.

Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation.

AIMS: Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST. METHODS AND RESULTS: Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+/-dP/dtmax), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +/-dP/dtmax were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 +/- 2% in the control group to 29 +/- 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 +/- 5%) was not different from the KO control group (53 +/- 5% of risk area, n = 4, P = NS). CONCLUSIONS: A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1-KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.

A sphingosine kinase 1 mutation sensitizes the myocardium to ischemia/reperfusion injury.

OBJECTIVE: Sphingosine kinase (SphK) is a key enzyme in the synthesis of sphingosine 1-phosphate (S1P), a bioactive sphingolipid. SphK is involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK1 in myocardial ischemia/reperfusion (IR) injury and IPC. METHODS AND RESULTS: Wild-type and SphK1 null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), +/-dP/dtmax, and LV end-diastolic pressure (EDP) between SphK1 mutant and wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S1P levels were decreased by 41%. SphK1 null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and +/-dP/dtmax decreased, LVEDP increased, and infarct size increased (n=6, P<0.05). Apoptosis was markedly enhanced in SphK1 mutant IR mouse hearts. IPC was cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK1 null hearts. There was no change in infarct size in the IPC+IR group compared to the IR group in the null hearts (50.1+/-5.0% vs 45.0+/-3.8%, n=6, P=NS). IPC remained ineffective in the null hearts even when the index ischemia time was shortened by 10 min. CONCLUSIONS: Deletion of the SphK1 gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These data provide the first genetic evidence that the SphK1-S1P pathway is a critical mediator of IPC and cell survival.

Low dose N, N-dimethylsphingosine is cardioprotective and activates cytosolic sphingosine kinase by a PKCepsilon dependent mechanism.

OBJECTIVE: N, N-Dimethylsphingosine (DMS) is recognized as an inhibitor of sphingosine kinase (SphK), a key enzyme responsible for the formation of sphingosine-1-phosphate (S1P). We previously showed that S1P was cardioprotective and that SphK was critical for myocardial ischemic preconditioning. Although DMS is an endogenous sphingolipid, its effect on cardiac function and cardioprotection at low concentration has not been studied. METHODS: In Langendorff-perfused wild-type and protein kinase C (PKC)epsilon-null mouse hearts, cardiac function, infarction size, and SphK activity were measured. RESULTS: Pretreatment with 0.3 microM and 1 microM DMS for 10 min protected against ischemia/reperfusion injury. Cardiac function (LVDP, +/-dP/dtmax) was improved and infarction size was reduced. The cardiac protection induced by DMS was abolished in PKCepsilon-null mouse hearts. Administration of 1 microM DMS ex vivo increased cytosolic SphK activity. This enhanced SphK activity was abolished in PKCepsilon-null mouse hearts. DMS also increased PKCepsilon translocation from the particulate to the cytosolic fraction with no effect on PKCalpha distribution. Co-immunoprecipitation showed that SphK1 interacted with PKCepsilon phosphorylated on Ser729. DMS also increased cytosolic Akt phosphorylation (Ser 473) and Akt translocation from a Triton-insoluble fraction to the cytosol. CONCLUSIONS: DMS has a biphasic effect on cardioprotection. Higher concentrations (10 microM) are inhibitory, whereas a low concentration (0.3 microM and 1 microM) of DMS protects murine hearts against ischemia/reperfusion injury. DMS activates SphK in the cytosol via a PKCepsilon dependent mechanism. The PKCepsilon-SphK-S1P-Akt pathway is involved in the cardiac protection induced by DMS.

Depletion of T lymphocytes ameliorates cardiac fibrosis in streptozotocin-induced diabetic cardiomyopathy.

T cell infiltration has been associated with increased coronary heart disease risk in patients with diabetes mellitus. Effect of modulation of T cell trafficking on diabetes-induced cardiac fibrosis has yet to be determined. Therefore, our aim was to investigate the circulatory T cell depletion-mediated cardioprotection in streptozotocin-induced diabetic cardiomyopathy. Fingolimod (FTY720), an immunomodulatory drug, was tested in wild-type (WT) C57BL/6 and recombination activating gene 1 (Rag1) knockout (KO) mice without mature lymphocytes in streptozotocin-induced type 1 diabetic model. FTY720 (0.3mg/kg/day) was administered intraperitoneally daily for the first 4weeks with interim 3weeks then resumed for another 4weeks in 11weeks study period. T lymphocyte counts, cardiac histology, function, and fibrosis were examined in diabetic both WT and KO mice. FTY720 reduced both CD4(+) and CD8(+) T cells in diabetic WT mice. FTY720-treated diabetic WT mouse myocardium showed reduction in CD3 T cell infiltration and decreased expression of S1P1 and TGF-ß1 in cardiac tissue. Fibrosis was reduced after FTY720 treatment in diabetic WT mice. Rag1 KO mice exhibited no CD4(+) and CD8(+) T cells in the blood and CD3 T cells in the heart. Diabetic Rag1 KO mouse hearts appeared no fibrosis and exhibited preserved myocardial contractility. FTY720-induced antifibrosis was abolished in diabetic Rag1 KO mice. These findings demonstrate that chronic administration with FTY720 induces lymphopenia and protects diabetic hearts in WT mice whereas FTY720 increases cardiac fibrosis and myocardial dysfunction in diabetic Rag1 KO mice without mature lymphocytes.

Sphingosine kinase activation mediates ischemic preconditioning in murine heart.

BACKGROUND: Phosphorylation of sphingosine by sphingosine kinase (SK) is the rate-limiting step in the cellular synthesis of sphingosine 1-phosphate (S1P). The monoganglioside GM1, which stimulates SK, is cardioprotective in part through increased generation of S1P that protects myocytes by diverse mechanisms. Because protein kinase C (PKC)epsilon activation is necessary for myocardial ischemic preconditioning (IPC) and PKC activators increase SK activity, we tested the hypothesis that SK may be a central mediator of IPC. METHODS AND RESULTS: In adult murine hearts, IPC sufficient to reduce infarct size significantly increased cardiac SK activity, induced translocation of SK protein from the cytosol to membranes, and enhanced cardiac myocyte survival. IPC did not increase SK activity in PKCepsilon-null mice. The SK antagonist N,N-dimethylsphingosine inhibited PKCepsilon activation and directly abolished the protective effects of IPC and the enhanced SK activity induced by IPC. CONCLUSIONS: These findings demonstrate that PKCepsilon is thus recruited by IPC and induces activation of SK that then mediates IPC-induced cardioprotection in murine heart.

Inhibition of PKC-θ preserves cardiac function and reduces fibrosis in streptozotocin-induced diabetic cardiomyopathy.

BACKGROUND AND PURPOSE: T-cell infiltration, interstitial fibrosis and cardiac dysfunction have been observed in diabetic patients with cardiovascular diseases. PKC-θ is crucial for the activation of mature T-cells. We hypothesized that inhibition of PKC-θ might protect diabetic hearts through inhibition of T-cell stimulation and maintenance of tight junction integrity. EXPERIMENTAL APPROACH: A model of type 1 diabetes was induced by streptozotocin (STZ) (50 mg kg(-1) for 5 days) in male C57BL/6J wild-type (WT) mice and Rag1 knockout (KO) mice which lack mature lymphocytes. A cell-permeable selective PKC-θ peptide inhibitor (PI) was administered i.p. (0.2 mg kg(-1) ·day(-1) ) for 4 weeks (first phase) and 2 weeks (second phase). At the end of the 11th week, cardiac contractile force was measured in isolated perfused hearts. Cardiac morphology and fibrosis were determined. Phosphorylation of PKC-θ at Tyr(358) , infiltrated T-cells and tight junction protein ZO-1 within the hearts were detected, using immunohistochemcial techniques. KEY RESULTS: PI did not affect high blood glucose level in both WT and Rag1 KO diabetic mice. Diabetes induced cardiac fibrosis in WT mice but not in Rag1 KO mice. PI attenuated cardiac fibrosis and improved cardiac contractility of WT diabetic hearts. PI decreased expression of phosphorylated PKC-θ, reduced the infiltration of T-cells and increased ZO-1 expression within WT diabetic hearts. CONCLUSION AND IMPLICATIONS: Inhibition of PKC-θ improves cardiac function and reduces cardiac fibrosis in WT mice with streptozotocin-induced diabetes. Mature T-cells play a key role in pathophysiology of diabetic cardiomyopathy.

N-terminal truncated intracellular matrix metalloproteinase-2 induces cardiomyocyte hypertrophy, inflammation and systolic heart failure.

Matrix metalloproteinase-2 (MMP-2) is increasingly recognized as a major contributor to progressive cardiac injury within the setting of ischemia-reperfusion injury and ischemic ventricular remodeling. A common feature of these conditions is an increase in oxidative stress, a process that engages multiple pro-inflammatory and innate immunity cascades. We recently reported on the identification and characterization of an intracellular isoform of MMP-2 generated by oxidative stress-mediated activation of an alternative promoter located within the first intron of the MMP-2 gene. Transcription from this site generates an N-terminal truncated 65 kDa isoform of MMP-2 (NTT-MMP-2) that lacks the secretory sequence and the inhibitory prodomain region. The NTT-MMP-2 isoform is intracellular, enzymatically active and localizes in part to mitochondria. Expression of the NTT-MMP-2 isoform triggers Nuclear Factor of Activated T-cell (NFAT) and NF-κB signaling with the expression of a highly defined innate immunity transcriptome, including Interleukin-6, MCP-1, IRF-7 and pro-apoptotic transcripts. To determine the functional significance of the NTT-MMP-2 isoform in vivo we generated cardiac-specific NTT-MMP-2 transgenic mice. These mice developed progressive cardiomyocyte and ventricular hypertrophy associated with systolic heart failure. Further, there was evidence for cardiomyocyte apoptosis and myocardial infiltration with mononuclear cells. The NTT-MMP-2 transgenic hearts also demonstrated more severe injury following ex vivo ischemia-reperfusion injury. We conclude that a novel intracellular MMP-2 isoform induced by oxidant stress directly contributes, in the absence of superimposed injury, to cardiomyocyte hypertrophy. inflammation, systolic heart failure and enhanced susceptibility to ischemia-reperfusion injury.

A sphingosine kinase form 2 knockout sensitizes mouse myocardium to ischemia/reoxygenation injury and diminishes responsiveness to ischemic preconditioning.

Sphingosine kinase (SphK) exhibits two isoforms, SphK1 and SphK2. Both forms catalyze the synthesis of sphingosine 1-phosphate (S1P), a sphingolipid involved in ischemic preconditioning (IPC). Since the ratio of SphK1:SphK2 changes dramatically with aging, it is important to assess the role of SphK2 in IR injury and IPC. Langendorff mouse hearts were subjected to IR (30 min equilibration, 50 min global ischemia, and 40 min reperfusion). IPC consisted of 2 min of ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), ± dP/dtmax, and heart rate between SphK2 null (KO) and wild-type (WT) hearts. In KO hearts, SphK2 activity was undetectable, and SphK1 activity was unchanged compared to WT. Total SphK activity was reduced by 53%. SphK2 KO hearts subjected to IR exhibited significantly more cardiac damage (37 ± 1% infarct size) compared with WT (28 ± 1% infarct size); postischemic recovery of LVDP was lower in KO hearts. IPC exerted cardioprotection in WT hearts. The protective effect of IPC against IR was diminished in KO hearts which had much higher infarction sizes (35 ± 2%) compared to the IPC/IR group in control hearts (12 ± 1%). Western analysis revealed that KO hearts had substantial levels of phosphorylated p38 which could predispose the heart to IR injury. Thus, deletion of the SphK2 gene sensitizes the myocardium to IR injury and diminishes the protective effect of IPC.

MnSOD in mouse heart: acute responses to ischemic preconditioning and ischemia-reperfusion injury.

Manganese superoxide dismutase (MnSOD) is one of the main antioxidant enzymes that protects the heart against ischemia-reperfusion (I/R) injury. Ischemic preconditioning (IPC) is a short period of ischemia-reperfusion that reduces subsequent prolonged I/R injury. Although MnSOD localizes in mitochondria, the immediate subcellular distribution of MnSOD in heart after IPC and I/R has not been studied. In a Langendorff mouse heart model, IPC significantly improved cardiac function and reduced the infarction size induced by I/R. Immunoblotting and double immunostaining in fresh preparations revealed that I/R resulted in an increase in cytosolic MnSOD content accompanied by the release of cytochrome c. In contrast, IPC increased mitochondrial MnSOD and reduced cytosolic MnSOD and cytochrome c release induced by I/R. We found that compared with freshly prepared fractions, the freeze-thaw approach results in mitochondrial integrity disruption and release of large amounts of MnSOD into the cytosol along with mitochondrial markers even in the absence of I/R. In contrast, fresh preparations exhibit early MnSOD release into the cytosol after I/R that is prevented by IPC and cyclosporin A administration.

Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKC epsilon knockout mouse hearts.

Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.