Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction. extracellular matrix; remodeling; T lymphocyte; interferon regulatory factor 1; toll-like receptor-9; lysyl oxidase LEFT VENTRICULAR REMODELING in response to pressure and/or volume overload is associated with myocardial hypertrophy, fibroblast hyperplasia, and increased concentrations of fibrillar collagen in the extracellular matrix. Recent studies have shown that pathological myocardial fibrosis may cause diastolic dysfunction and heart failure (24), whereas myocyte hypertrophy in the absence of fibrosis is considered physiological remodeling (22, 46) if the mass-to-volume ratio is preserved (5). In this study, we investigated whether interleukin (IL)-18 regulates the gene and protein expression of the matricellular protein osteopontin (OPN), a component and regulator of cardiac extracellular matrix fibrillar collagen concentrations.As reviewed by Okamoto (26), OPN also plays a critical role in the regulation of cellular proliferation and differentiation, interstitial fibrosis, arteriosclerosis, and angiogenesis. Although OPN is ubiquitously expressed in many tissues and is increased during stress-induced cardiac remodeling, it participates as a cytokine or humoral factor in the in...

Aberrant concentrations of cardiac extracellular matrix (ECM) fibrillar collagen crosslinking have been proposed to be an underlying cause of cardiac diastolic dysfunction however the role of the adaptive immune system in this process has yet to be investigated. Fibrillar collagen crosslinking is a product of the enzymatic activities of lysyl oxidase (LOX and LOXL-3) released by the cardiac fibroblast and possibly cardiac myocytes. Our hypothesis is that stimulation of the TH1 lymphocytes activates lysyl oxidase mediated ECM crosslinking and thereby alter left ventricular function. Threemonth old C57BL/J female mice were treated with selective TH1 lymphocyte inducers -T-cell receptor Vβ peptides (TCR). After 6 weeks, candidate gene expression, tissue enzymatic activity, ECM composition, and left ventricular mechanics were quantified. Lymphocyte gene expression and cytokine assay revealed TH1 immune polarization with TCR administration which was associated with a 2.6-fold and 3.1-fold increase of LOX and LOXL3 gene expression, respectively, and a 55% increase in cardiac LOX enzymatic activity. The ECM cross-linked fibrillar collagen increased by 95% when compared with the control. Concurrently, there was a 33% increased ventricular stiffness, decreased cardiac output, and normal ejection fraction. These data implicate the TH1 lymphocyte in the pathogenesis of diastolic dysfunction which has potential clinical application in the pathogenesis of diastolic heart failure.

Metabolic syndrome (MetS) represents an increased risk of cardiovascular disease. Although its individual components adversely affect cardiac structure and function, the extent to which multiple components of MetS affect the cardiac extracellular matrix (ECM) has not been well characterized. Lysyl oxidase (LOX) is one of the cardiac ECM-modifying enzymes that catalyze the formation of collagen cross-linking. Our objective was to define the effect of diet-induced MetS on the LOX enzyme. MetS was induced in male C57BL/6 mice by administrating a high-fat, high-simple carbohydrate diet for 6 mo. Gene expression was determined by real-time PCR. The cardiac protein expression and enzymatic activity of LOX were measured. The severity of fibrosis was assessed by histology and hydroxylproline assay. Cardiac diastolic function was assessed by in vivo analysis of the pressure-volume relationship. LOX, matrix metalloproteinases, and their tissue inhibitors were analyzed, and of these three, LOX was most significantly changed in the MetS mice. Despite the blunted gene expression of LOX isoforms, MetS mice demonstrated a significant upregulation of bone morphogenetic protein-1. Correspondingly, there was an increase in the ratio of protein expression of mature to proenzyme LOX by 25.9%, enhanced LOX activity by 50.0%, and increased cardiac cross-linked collagen compared with the controls. This fibrotic response coincided with a marked increase in end-diastolic pressure, increased left ventricular stiffness, and impaired diastolic filling pattern. Our data signify that diet-induced MetS alters the remodeling enzymes, mainly LOX, thereby altering ECM structure by increasing the amount of cross-linking and inducing diastolic dysfunction.

During cardiopulmonary bypass (CPB), perfusion at tepid temperatures (33-35 °C) is recommended to avoid high temperature cerebral hyperthermia during and after the operation. However, the ideal temperature for uncomplicated adult cardiac surgery is an unsettled question. Typically, the heat exchanger maximum temperature is monitored between 40-42 °C to prevent denaturation of plasma proteins, but studies have not been performed to make these conclusions. Therefore, our hypothesis was to determine the temperature in which blood plasma protein degradation occurs after 2 hours of heat exposure. As a result, blood plasma proteins were exposed to heat in the 37-50 °C range for 2 hours. Plasma protein samples were loaded onto an 8-12% gradient gel for SDS-PAGE and low molecular weight plasma protein degradation was detected with graded heat exposure. Protein degradation was first detected between 43-45 °C of heat exposure. This study supports the practice of monitoring the heat exchanger between 40-42 °C to prevent denaturation of plasma proteins.

Left ventricular diastolic dysfunction is an important predictor of prognosis and mortality of heart failure. Increased left ventricular stiffness can be associated with excessive myocardial fibrosis and increased cross-linked collagen by the enzyme lysyl oxidase (LOX). These cardiac extracellular matrix (ECM) remodeling processes are affected by T-lymphocyte function and phenotype. We sought to examine the role of T lymphocytes in myocardial LOX regulation in diet-induced fibrotic hearts. Female SCID mice, devoid of functional T lymphocytes, and wild-type (WT) C57BL/6 were treated with a high-fat high-simple carbohydrate (HFHSC) diet for 12 months. HFHSC-fed WT mice demonstrated a significant increase in the catalytic activity of myocardial LOX compared with respective controls. These changes coincided with a marked increase in ECM collagen cross-linking and impaired diastolic filling pattern. However, induction of LOX was minimal in the SCID mice compared with the WT group. Correspondingly fibrillar cross-linked collagen concentrations and diastolic dysfunction were less prominent in the SCID mice compared with the WT group. Our results suggest a role for T lymphocytes in this dietary induction of diastolic dysfunction through modulation of LOX-dependent collagen maturation.

Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.

The adrenal steroid hormone dehydroepiandrosterone (DHEA) and its sulfated derivative [DHEA(S)] have been extensively studied for their potential anti-aging effects. Associated with aging, DHEA levels decline in humans, whereas other adrenal hormones remain unchanged, suggesting that DHEA may be important in the aging process. However, the effect of DHEA(S) supplementation on cardiac function in the aged has not been investigated. Therefore, we administered to young and old female mice a 60-day treatment with exogenous DHEA(S) at a dose of 0.1 mg/ml in the drinking water and compared the effects on left ventricular diastolic function and the myocardial extracellular matrix composition. The left ventricular stiffness (beta) was 0.30 +/- 0.06 mmHg/mul in the older control mice compared with 0.17 +/- 0.02 mmHg/mul in young control mice. Treatment with DHEA(S) decreased left ventricular stiffness to 0.12 +/- 0.03 mmHg/mul in the older mice and increased left ventricular stiffness to 0.27 +/- 0.04 mmHg/mul in young mice. The mechanism for the DHEA(S)-induced changes in diastolic function appeared to be associated with altered matrix metalloproteinase activity and the percentage of collagen cross-linking. We conclude that exogenous DHEA(S) supplementation is capable of reversing the left ventricular stiffness and fibrosis that accompanies aging, with a paradoxical increased ventricular stiffness in young mice.

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