Cytokines In Cardiomyocytes Research Articles

MECHANISMS OF CARDIAC DYSFUNCTION IN SEPSIS.

Studies in animal models of sepsis have elucidated an intricate network of signaling pathways that lead to the dysregulation of myocardial Ca 2+ handling and subsequently to a decrease in cardiac contractile force, in a sex- and model-dependent manner. After challenge with a lethal dose of LPS, male animals show a decrease in cellular Ca 2+ transients (ΔCa i ), with intact myofilament function, whereas female animals show myofilament dysfunction, with intact ΔCa i . Male mice challenged with a low, nonlethal dose of LPS also develop myofilament desensitization, with intact ΔCa i . In the cecal ligation and puncture (CLP) model, the causative mechanisms seem similar to those in the LPS model in male mice and are unknown in female subjects. ΔCa i decrease in male mice is primarily due to redox-dependent inhibition of sarco/endoplasmic reticulum Ca 2+ ATP-ase (SERCA). Reactive oxygen species (ROS) are overproduced by dysregulated mitochondria and the enzymes NADPH/NADH oxidase, cyclooxygenase, and xanthine oxidase. In addition to inhibiting SERCA, ROS amplify cardiomyocyte cytokine production and mitochondrial dysfunction, making the process self-propagating. In contrast, female animals may exhibit a natural redox resilience. Myofilament dysfunction is due to hyperphosphorylation of troponin I, troponin T cleavage by caspase-3, and overproduction of cGMP by NO-activated soluble guanylate cyclase. Depleted, dysfunctional, or uncoupled mitochondria likely synthesize less ATP in both sexes, but the role of energy deficit is not clear. NO produced by NO synthase (NOS)-3 and mitochondrial NOSs, protein kinases and phosphatases, the processes of autophagy and sarco/endoplasmic reticulum stress, and β-adrenergic insensitivity may also play currently uncertain roles.

Absent in Melanoma 2 (AIM2) limits pro-inflammatory cytokine transcription in cardiomyocytes by inhibiting STAT1 phosphorylation.

Interferon (IFN)-γ is highly upregulated during heart inflammation and enhances the production of pro-inflammatory cytokines. Absent in Melanoma 2 (AIM2) is an IFN-inducible protein implicated as a component of the inflammasome. Here we seek to determine the role of AIM2 during inflammation in cardiac cells. We found that the presence of AIM2, but not of the other inflammasome components Nod-like receptor (NLR) NLRP3 or NLRC4, specifically limited the transcription of the pro-inflammatory cytokines interleukin (IL)-6, IP-10, and tumor necrosis factor (TNF)-α in HL-1 mouse cardiomyocytes stimulated with IFN-γ and lipopolysaccharides (LPS). Similarly, AIM2 reduced pro-inflammatory cytokine transcription in primary mouse neonatal cardiomyocytes (MNC), but not in primary mouse neonatal cardiac fibroblasts (MNF). Interestingly, AIM2-dependent reduction of pro-inflammatory cytokines in cardiomyocytes was independent of Caspase-1. Mechanistically, AIM2 reduced pro-inflammatory cytokine transcription in cardiomyocytes by interacting with and inhibiting the phosphorylation of STAT1. In AIM2-depleted cardiomyocytes, increased STAT1 phosphorylation enhanced the NF-κB pathway by promoting NF-κB p65 phosphorylation and acetylation. These results show for the first time that AIM2 plays an important anti-inflammatory, yet inflammasome-independent function in cardiomyocytes. Our findings will help to further understand how the various heart cell types differently react to inflammatory stimuli.

The inhibition of N-glycosylation of glycoprotein 130 molecule abolishes STAT3 activation by IL-6 family cytokines in cultured cardiac myocytes.

Interleukin-6 (IL-6) family cytokines play important roles in cardioprotection against pathological stresses. IL-6 cytokines bind to their specific receptors and activate glycoprotein 130 (gp130), a common receptor, followed by further activation of STAT3 and extracellular signal-regulated kinase (ERK)1/2 through janus kinases (JAKs); however the importance of glycosylation of gp130 remains to be elucidated in cardiac myocytes. In this study, we examined the biological significance of gp130 glycosylation using tunicamycin (Tm), an inhibitor of enzyme involved in N-linked glycosylation. In cardiomyocytes, the treatment with Tm completely replaced the glycosylated form of gp130 with its unglycosylated one. Tm treatment inhibited leukemia inhibitory factor (LIF)-mediated activation of STAT3 and ERK1/2. Similarly, IL-11 failed to activate STAT3 and ERK1/2 in the presence of Tm. Interestingly, Tm inhibited the activation of JAKs 1 and 2, without influencing the expression of suppressor of cytokine signalings (SOCSs) and protein-tyrosine phosphatase 1B (PTP1B), which are endogenous inhibitors of JAKs. To exclude the possibility that Tm blocks LIF and IL-11 signals by inhibiting the glycosylation of their specific receptors, we investigated whether the stimulation with IL-6 plus soluble IL-6 receptor (sIL-6R) could transduce their signals in Tm-treated cardiomyocytes and found that this stimulation was unable to activate the downstream signals. Collectively, these findings indicate that glycosylation of gp130 is essential for signal transduction of IL-6 family cytokines in cardiomyocytes.

Future prospects of Qiliqiangxin on heart failure: epigenetic regulation of regeneration

Qiliqiangxin (QL), a traditional Chinese medicine, was approved by China Food and Drug Administration for the treatment of heart failure in 2004 (Li et al., 2013). Our recent multicenter, randomized, double-blind, parallel-group, placebo-controlled study of the effects of QL capsules in patients with chronic heart failure shows that on a background of standard treatment, QL capsules further decreases the levels of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) during 12 weeks of treatment (Li et al., 2013). However, the underlying mechanism is still unclear. Recent progress in studying QL on improving exercise tolerance and patients' quality of life highlighted that QL may be through a variety of biological mechanisms to exert its beneficial effects. One possible mechanism may be the regulation of the balance between pro-inflammatory and anti-inflammatory cytokines in cardiomyocytes (Xiao et al., 2009). In addition, it may down-regulate the cardiac chymase signaling pathway and chymase-mediated AngII production (Zhao et al., 2012). Moreover, it may affect cardiomyocyte death and proliferation, leading to improved cardiac remodeling and cardiac function (Zou et al., 2012). Traditionally, the adult mammalian heart is regarded as a post-mitotic organ without any regenerative capacity (Rosenzweig, 2012). Accumulating evidence shows that cardiomyocytes in adult mammals actually retain a limited ability to proliferation (Rosenzweig, 2012). Recently, a study shows that exercise induce physiological hypertrophy featured by cardiomyocyte hypertrophy and proliferation. The cardiomyocyte proliferation is mediated by the reduction of C/EBPs and a linked increased of CBP/p300-interacting transactivator with ED-rich carboxyterminal domain 4 (CITED4) (Bostrom et al., 2010). Interestingly, QL has been reported to decrease C/EBPs and also increase CITED4 at 4 weeks after transverse aorta constriction induced cardiac hypertrophy, remodeling and dysfunction in mice (Zou et al., 2012). The changes of C/EBPs and CITED4 are paralleled with enhanced proliferation of cardiomyocytes (Zou et al., 2012). Moreover, our recent study also proves that Huangqi (one of the major components of QL) improves cardiac function after acute myocardial infarction in mice by regulating mTORC1 signaling pathway, which also medicates cell proliferation (Wu et al., 2013). In conclusion, we think QL may mimic a phenotype of physiological hypertrophy and induce heart regeneration, which is beneficial for heart failure.

Bactericidal/permeability increasing protein attenuates the myocardial inflammation/dysfunction that occurs with burn complicated by subsequent infection

Intubation and mechanical ventilation after burn contribute to pneumonia-related infection. Although postburn presence or absence of endotoxin has been described, inactivation of Toll-like receptor 4 signaling has been shown to improve postburn organ function, suggesting that LPS participates in burn-related susceptibility to infection. We hypothesized that bactericidal/permeability-increasing protein (rBPI) given postburn would attenuate myocardial inflammation/dysfunction associated with postburn septic challenge given 7 days postburn. Rats were given burn over 40% total body surface area, lactated Ringer 4 ml.kg(-1).% burn(-1); burns received either vehicle or rBPI, 1 mg.kg(-1).h(-1) for 48 h postburn. Postburn day 7, subgroups of burns and shams were given intratracheal Klebsiella pneumoniae, 4 x 10(6) CFU to produce burn complicated by sepsis; additional sham and burn subgroups received intratracheal vehicle to produce sham sepsis. Vehicle-treated groups: 1) sham burn + sham sepsis 2) sham burn + sepsis, 3) burn + sham sepsis, 4) burn + sepsis. rBPI-treated groups: 5) sham burn + sham sepsis, 6) sham burn + sepsis, 7) burn + sham sepsis, 8) burn + sepsis. Cardiomyocyte cytokine secretion and myocardial function were studied 24 h after septic challenge, postburn day 8. Pneumonia-related infection 8 days after vehicle-treated burn produced myocyte cytokine secretion (pg/ml), indicated by increased myocyte TNF-alpha, 549 +/- 46; IL-1beta, 50 +/- 8; IL-6, 286 +/- 3 levels compared with levels in sham myocytes (TNF-alpha, 88 +/- 11; IL-1beta, 7 +/- 1; IL-6, 74 +/- 10; P < 0.05). Contractile dysfunction was evident from lower left ventricular pressure +/-dP/dt values in this group compared with sham. rBPI attenuated myocyte cytokine responses to septic challenge and improved contractile function, suggesting that burn-related mobilization of microbial-like products contribute to postburn susceptibility to infection.

Effects of burn injury on myocardial signaling and cytokine secretion: possible role of PKC

This study examined the effects of major burn injury on the cellular distribution of several PKC isoforms in adult rat hearts and examined the hypothesis that PKC plays a regulatory role in cardiomyocyte cytokine secretion. Burn trauma was given over 40% total body surface area in Sprague-Dawley rats. An in vitro model of burn injury included addition of burn serum, 10% by volume, to primary cardiomyocyte cultures (collagen perfusion). In vivo burn injury produced redistribution of PKCdelta, PKCepsilon, and PKCalpha from the cytosol (soluble) to the membrane (particulate) component of the myocardium. This activation of the PKC isoforms was evident 2 h after burn injury and progressively increased over 24 h postburn. Addition of burn serum to isolated myocytes produced similar PKC isoform redistribution from the soluble to the particulate compartment, promoted myocyte Ca2+ and Na+ loading, and promoted robust myocyte secretion of inflammatory cytokines similar to that reported after in vivo burn injury. Pretreating cardiomyocytes with either calphostin or PKCepsilon inhibitory peptide, a potent inhibitor of PKCepsilon, prevented burn serum-related redistribution of the PKCepsilon isoform and prevented burn serum-related cardiomyocyte secretion of TNF-alpha, IL-1beta, IL-6, and IL-10. These data suggest that the PKCepsilon isoform plays a pivotal role in myocardial inflammatory response to injury, altering cardiac function by modulating cardiomyocyte inflammatory cytokine response to injury.

Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis

Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors,and not on gene deletion. Here we used ROCK1knockout (ROCK1-/-) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1-/- mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1-/- mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFbeta2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of Gq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli.

Effect of Aspiration Pneumonia-Induced Sepsis on Post-Burn Cardiac Inflammation and Function in Mice

Numerous studies have found that burn injury alters immune function, predisposing the subject to infectious complications. We developed a mouse model of burn injury complicated by either gram-positive or gram-negative infection and hypothesized that post-burn infection would exacerbate the myocardial cytokine responses and contractile dysfunction characteristic of either sepsis alone or burn alone. Adult C57 BL6 mice were given burn injury over 40% of the total body surface area and conventional fluid resuscitation (lactated Ringer's solution, 4 mL/kg/% burn) followed on day 7 by intratracheal administration of 1 x 10(5) cfu of either Streptococcus pneumoniae or Klebsiella pneumoniae or saline. Mice received fluid resuscitation (2 mL of lactated Ringer's intraperitoneally) again after bacterial challenge. Cardiomyocyte cytokine secretion and the contractile function of isolated hearts (Langendorff perfusion) were examined in vitro 24 h after bacterial challenge. Infectious challenge seven days after burn injury exaggerated the inflammatory cytokine responses over those observed with either burn alone or gram-positive or gram-negative infection alone (tumor necrosis factor-alpha: sham, 72 +/- 9 pg/mL; burn alone, 176 +/- 6 pg/mL, Klebsiella pneumoniae alone, 337 +/- 8 pg/mL; Streptococcus pneumoniae alone, 184 +/- 2 pg/mL; burn + Klebsiella, 476 +/- 14 pg/mL; burn + Streptococcus, 351 +/- 6 pg/mL). Myocardial contractile depression was evident in the burn alone, infection alone, and burn plus infection groups, regardless of the organism selected to produce pneumonia-related sepsis. Gram-negative or gram-positive infection exacerbated the myocardial inflammation seen with burn alone or infection alone. The availability of a mouse model of burn injury complicated by pneumonia-related sepsis will allow use of genetically engineered mice to examine further the mechanisms by which burn injury increases susceptibility to infection.

CARDIOMYOCYTE FUNCTION AFTER BURN INJURY AND LIPOPOLYSACCHARIDE EXPOSURE: SINGLE-CELL CONTRACTION ANALYSIS AND CYTOKINE SECRETION PROFILE

A component of multiorgan dysfunction in burned patients is heart failure. Burn trauma induces cytokine synthesis of interleukin (IL) 1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) which can negatively impact cardiac function. Infectious complications are common following severe burn injury. We hypothesized that burn injury and lipopolysaccharide (LPS) exposure independently influence peak cardiomyocyte contraction and cytokine secretion. Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 1, 6, 12, and 24 h after burn, cardiomyocytes were isolated and incubated with increasing LPS doses. Peak sarcomere shortening and contractile velocity parameters were recorded using a variable-rate video camera with sarcomere length detection software. Supernatants were assayed for IL-1beta, IL-6, and TNF-alpha by ELISA. Peak sarcomere shortening was decreased in the burn group at 1, 6, 12, and 24 h after burn. IL-1beta, IL-6, and TNF-alpha levels were increased in cardiomyocytes isolated 1 h after burn compared with sham controls, but returned to sham levels at 6, 12, and 24 h after burn. LPS exposure caused dose-dependent decreases in sarcomere shortening in sham and burn animals. LPS exposure did not produce increased cardiomyocyte cytokine expression. Burn injury diminished peak sarcomere shortening. Whereas exposure to LPS did not have an effect on cardiomyocyte cytokine expression, LPS significantly inhibited sarcomere shortening in a dose-dependent fashion. Combined burn and LPS exposure inhibited sarcomere shortening more than each alone. These results demonstrate that LPS exposure and burn injury independently decrease peak cardiac shortening. These decreases did not directly correlate with the levels of cytokines released in response to each stressor.

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein.

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.

Hypertonic saline-dextran suppresses burn-related cytokine secretion by cardiomyocytes.

Whereas hypertonic saline-dextran (HSD, 7.5% NaCl in 6% D70) improves cardiac contractile function after burn trauma, the mechanisms of HSD-related cardioprotection remain unclear. We recently showed that cardiomyocytes secrete tumor necrosis factor-alpha (TNF-alpha), a response that was enhanced by burn trauma. This study addressed the question: does HSD modulate cardiac contraction/relaxation by altering cardiomyocyte TNF-alpha secretion? Wistar-Furth rats (325 g) were given a burn injury over 40% of the total body surface area and were then randomized to receive a bolus of either isotonic saline or HSD (4 ml/kg, n = 14 rats/group). Sham burn rats were given either isotonic saline or HSD (n = 14 rats/group) to provide appropriate controls for the two burn groups. Hearts were isolated 24 h postburn for either Langendorff perfusion (n = 8 hearts/group) or to prepare cardiomyocytes (n = 6 hearts/group). Myocytes were stimulated with lipopolysaccharide (LPS) (0, 10, 25, or 50 microg for 18 h) to measure cytokine secretion. Burn trauma increased myocyte TNF-alpha and interleukin-1 beta and -6 secretion, exacerbated cytokine response to LPS stimulus, and impaired cardiac contraction. HSD treatment of burns decreased cardiomyocyte cytokine secretion, decreased responsiveness to LPS challenge with regard to cytokine secretion, and improved ventricular function. These data suggest that HSD mediates cardioprotection after burn trauma, in part, by downregulating cardiomyocyte secretion of inflammatory cytokines.