Scleraxis and fibrosis in the pressure-overloaded heart.

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

Spermidine/spermine N1-acetyltransferase-1 as a diagnostic biomarker in human cancer.

AIM: SSAT-1 is an enzyme that plays a critical role in cell growth. Amantadine, a FDA-approved antiviral drug, is a substrate for SSAT-1. The utility of amantadine as an agent to demonstrate elevated SSAT-1 activity linked to cancer was conducted. RESULTS: High levels of SSAT-1 expression were measured in tumor human cell lines, and in breast, prostate and lung tumor tissue. An increase in the urinary levels of acetylated amantadine in cancer patients was observed. CONCLUSION: Increases in SSAT-1 contents in tumor tissue could be of value in targeting cancers with high SSAT-1 expression for confirmation/quantification. The high levels of acetylated amantadine could be used as a simple and useful screening test for the presence of cancer.

Role of scleraxis in mechanical stretch-mediated regulation of cardiac myofibroblast phenotype.

The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-ß, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.

The transcription factor scleraxis is a critical regulator of cardiac fibroblast phenotype.

BACKGROUND: Resident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis. RESULTS: Using explanted primary cardiac fibroblasts in gain- and loss-of-function studies, we found that scleraxis critically controls cardiac fibroblast/myofibroblast phenotype by direct transcriptional regulation of myriad genes that effectively define these cells, including extracellular matrix components and α-smooth muscle actin. Scleraxis furthermore potentiated the TGFß/Smad3 signaling pathway, a key regulator of myofibroblast conversion, by facilitating transcription complex formation. While scleraxis promoted fibroblast to myofibroblast conversion, loss of scleraxis attenuated myofibroblast function and gene expression. These results were confirmed in scleraxis knockout mice, which were cardiac matrix-deficient and lost ~50% of their complement of cardiac fibroblasts, with evidence of impaired epithelial-to-mesenchymal transition (EMT). Scleraxis directly transactivated several EMT marker genes, and was sufficient to induce mesenchymal/fibroblast phenotype conversion of A549 epithelial cells. Conversely, loss of scleraxis attenuated TGFß-induced EMT marker expression. CONCLUSIONS: Our results demonstrate that scleraxis is a novel and potent regulator of cellular progression along the continuum culminating in the cardiac myofibroblast phenotype. Scleraxis was both sufficient to drive conversion, and required for full conversion to occur. Scleraxis fulfills this role by direct transcriptional regulation of key target genes, and by facilitating TGFß/Smad signaling. Given the key role of fibroblast to myofibroblast conversion in fibrotic diseases in the heart and other tissue types, scleraxis may be an important target for therapeutic development.

Effects of amino acid supplementation on myocardial cell damage and cardiac function in diabetes.

Although amino acid deficiencies are known to occur in diabetes patients and are considered to contribute to the occurrence of cardiomyopathy, the mechanisms of the impact of the restoration of amino acids on improved cardiac function are not completely understood. Accordingly, the present study was conducted to examine the beneficial effects of dietary supplementation of taurine, arginine and carnitine, individually or in combination, in an experimental model of chronic diabetes. For inducing diabetes, rats received a single injection of streptozotocin (65 mg/kg body weight). Experimental animals were treated (by oral gavage) daily for three weeks with amino acids before the induction of diabetes; this treatment was continued for an additional eight-week period. Diabetes was observed to induce cardiac dysfunction, myocardial cell damage, and changes in plasma glucose and lipid levels. Treatment of diabetic animals with taurine, unlike carnitine or arginine, attenuated alterations in cardiac function, as evidenced by echocardiography and in vivo catheterization techniques. Taurine, carnitine and arginine, individually or in combination, attenuated diabetes-induced cell damage as revealed by electron microscopy. While carnitine alone reduced plasma levels of triglycerides with an increase in high-density lipoprotein cholesterol, none of the amino acids, alone or in combination, had an effect on myocardial glycogen content, lipid accumulation or hyperglycemia. These results suggest that dietary supplementation of taurine attenuates diabetes-induced changes in cardiac contractile function and ultrastructure without any alterations in plasma lipid and glucose levels.

Alterations in the expression of myocardial calcium cycling genes in rats fed a low protein diet in utero.

An adverse environmental experience of the growing fetus leads to permanent changes in the structure and contractile function of the heart; however, the mechanisms are incompletely understood. To examine if a maternal low protein (LP) diet can modulate the gene and protein expression of the Ca(2+)-cycling proteins in the neonatal heart, we employed a rat model in which pregnant dams were fed diets containing either 180 (normal) or 90 g (low) casein/kg diet for 2 weeks before mating and throughout pregnancy. A significant reduction in the L-type Ca(2+)-channel mRNA level in the LP group was detected at 1, 7, and 14 days of age. Although ryanodine receptor (RyR) mRNA levels progressively declined in the aging heart in both groups, the RyR mRNA levels were consistently higher in the LP group. A reduction in RyR protein content was seen only in the hearts of the LP group at 7 days of age. The Na(+)-Ca(2+)-exchanger (NCX) mRNA level was also markedly increased at all ages. Although an increase in sarco(endo)plasmic reticulum ATPase 2a (SERCA) 2a mRNA was only detected in the LP group at 7 days of age, corresponding protein level was depressed. On the other hand, an initial decrease (at 1 day of age) followed by an increase (at 14 and 28 days of age) in phospholamban (PLB) mRNA levels was detected. Although PLB protein level was also depressed at 1 day of age in the LP group, a marked increase was seen at 7 days of age. Moreover, the ratio of serine 16 and threonine 17 phosphorylated PLB to non-phosphorylated PLB was reduced at 7 days of age in the hearts of offspring of the LP group. These data suggest that maternal LP diet can induce alterations in the gene expression and protein levels of the Ca(2+)-cycling proteins in the neonatal heart.

Gender differences in the modulation of cardiac gene expression by dietary conjugated linoleic acid isomers.

In an earlier study, we showed that dietary conjugated linoleic acid (CLA) isomers can exert differential effects on heart function in male and female rats, but the underlying mechanisms for these actions are not known. Cardiomyocyte Ca2+ cycling is a key event in normal cardiac contractile function and defects in Ca2+ cycling are associated with cardiac dysfunction and heart disease. We therefore hypothesized that abnormalities in the sarcolemmal (SL) and sarcoplasmic reticulum (SR)-mediated regulation of intracellular Ca2+ contribute to altered cardiac contractile function of male and female rats owing to dietary CLA isomers. Healthy male and female Sprague-Dawley rats were fed different CLA isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. We determined the mRNA levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 2a, ryanodine receptor, phospholamban, calsequestrin, Na+-Ca2+-exchanger (NCX), and L-type Ca2+ channel in the left ventricle (LV) by RT-PCR. The SR function was assessed by measurement of Ca2+-uptake and -release. Significant gender differences were seen in the LV NCX, L-type Ca2+ channel, and ryanodine receptor mRNA expression levels in control male and female rats. Dietary CLA isomers in the various forms induced changes in the mRNA levels of SERCA 2a, NCX, and L-type Ca2+ channel in the LV of both male and female hearts. Whereas protein contents of the Ca2+ cycling proteins were altered, changes in SR Ca2+-uptake and -release were also detected in both male and female rats in response to dietary CLA. The results of this study demonstrate that long-term dietary supplementation can modulate cardiac gene expression and SR function in a gender-related manner and may, in part, contribute to altered cardiac contractility.

Gender differences in the cardiac response to dietary conjugated linoleic acid isomers.

The present study was undertaken to assess the heart function, by the in vivo catheterization technique, of healthy male and female Sprague-Dawley rats fed different conjugated linoleic acid (CLA) isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. Whereas the triglyceride form of the CLA isomer mix lowered the heart rate, the rate of contraction (+dP/dt) and rate of relaxation (-dP/dt), systolic and diastolic pressures, mean arterial pressure, and the left ventricular systolic pressure were higher in male rats as compared with all the other dietary groups. In contrast, there were no significant effects in the cardiac function of the female rats in response to the CLA isomer mix in triglyceride form. Whereas the heart rate, +dP/dt, and left ventricular systolic pressure were lower in male rats fed the t10,c12 CLA isomer alone, the heart rate of the female rats was higher, but the systolic pressure, +dP/dt, and mean arterial pressure were lower compared with the control group. Also, the left ventricular end-diastolic pressure was specifically higher in the female rats in response to free fatty acids-containing CLA mix. Furthermore, an additive effect of the free fatty acids-containing CLA mix was seen in the +dP/dt and -dP/dt of female rats compared with the control group. These results indicate that CLA isomers exert differential effects on heart function and suggest the need for a complete evaluation of the benefits, interactions, and potential side effects of each isomer.

Losartan attenuates phospholipase C isozyme gene expression in hypertrophied hearts due to volume overload.

Because the left ventricular (LV) hypertrophy due to volume overload induced by arteriovenous (AV) shunt was associated with an increase in phospholipase C (PLC) isozyme mRNA levels, PLC is considered to be involved in the development of cardiac hypertrophy. Since the renin-angiotensin system (RAS) is activated in cardiac hypertrophy, the role of RAS in the stimulation of PLC isozyme gene expression in hypertrophied heart was investigated by inducing AV shunt in Sprague-Dawley rats. The animals were treated with or without losartan (20 mg/kg, daily) for 3 days as well as 1, 2 and 4 weeks, and atria, right ventricle (RV) and LV were used for analysis. The increased muscle mass as well as the mRNA levels for PLC beta1 and beta3 in atria and RV, unlike PLC beta3 gene expression in LV, at 3 days of AVshunt were attenuated by losartan. The increased gene expression for PLC beta1 at 2 weeks in atria, at 1 and 4 weeks in RV, and at 2 and 4 weeks in LV was also depressed by losartan treatment. Likewise, the elevated mRNA levels for PLC beta3 in RV at 1 week and in LVat 4 weeks of cardiac hypertrophy were decreased by losartan. On the other hand, the increased levels of mRNA for PLC gamma1 in RV and LV at 2 and 4 weeks of inducing hypertrophy, unlike in atria at 4 weeks were not attenuated by losartan treatment. While the increased mRNA level for PLC delta1 in LV was reduced by losartan, gene expression for PLC delta1 was unaltered in atria and decreased in RV at 3 days of inducing AV shunt. These results suggest that changes in PLC isozyme gene expression were chamber specific and time-dependent upon inducing cardiac hypertrophy due to AV shunt. Furthermore, partial attenuation of the increased gene expression for some of the PLC isozymes and no effect of losartan on others indicate that both RAS dependent and independent mechanisms may be involved in hypertrophied hearts due to volume overload.

Phospholipid profile of developing heart of rats exposed to low-protein diet in pregnancy.

Although the myocardial phospholipid and fatty acid content have profound effects on the heart function, very little information is available on the effects of restricted maternal protein intake during pregnancy on the phospholipid profile and fatty acid content of the developing heart. The present study was therefore undertaken to examine the effect of pregnant dams fed diets containing either 180 (normal) or 90 (low) g/kg casein diet for 2 wk before mating and throughout pregnancy on myocardial phospholipid and fatty acid content of male offspring. Whereas no changes in phosphatidylcholine and phosphatidylethanolamine were detected, increases in lysophosphatidylcholine, phosphatidylserine, and sphingomyelin were seen in the hearts of offspring in the low-protein (LP) group. Analysis of cardiac fatty acids revealed that although the saturated fatty acid (myristate, palmitate, and stearate) levels were significantly reduced, the unsaturated fatty acid (linoleate, arachidonate, and decosahexanoate) levels were significantly increased in the developing heart in the LP group. Furthermore, assessment of nuclear transcription factors involved in regulation of cardiac metabolism revealed a decrease in myocyte enhancer factor-2C mRNA levels in the LP group, whereas an increase in the mRNA amount of peroxisome proliferator-activated receptor-alpha was observed in this group. These results demonstrate that maternal LP diet can induce changes in the phospholipid profile and fatty acid content of the developing heart, which may have implications for metabolism of the neonatal heart.

Prenatal exposure to maternal undernutrition induces adult cardiac dysfunction.

An adverse environmental experience of the growing fetus may lead to permanent changes in the structure and function of organs that may predispose the individual to chronic diseases in later life; however, nothing is known about the occurrence and mechanisms of heart failure. We employed a rat model in which pregnant dams were fed diets containing either 180 g (normal) or 90 g (low) casein/kg for 2 weeks before mating and throughout pregnancy. The ejection fraction (EF) of the pups exposed to the low-protein (LP) diet was severely depressed in the first 2 weeks of life and was associated with an increase in cardiomyocyte apoptosis. This early depressed cardiac function was followed by progressive recovery and normalization of the EF of the offspring in the LP group. The left ventricular (LV) internal diameters were increased between 24 h and 84 d (12 weeks) of age in the LP-exposed group. Although between 3 d and 2 weeks of age the LV wall of the heart in the LP group was thinner, a progressive increase in LV wall thickness was seen. At 40 weeks of age, although the EF was normal, a two-fold elevation in LV end-diastolic pressure, reduced cardiac output, decreased maximum rates of contraction and relaxation, and reduced mean arterial pressure were observed. Our findings demonstrate that exposure of the developing fetus to a maternal LP diet programs cardiac dysfunction in the offspring in later life.

Defective sarcolemmal phospholipase C signaling in diabetic cardiomyopathy.

Phospholipase C (PLC) activity is known to influence cardiac function. This study was undertaken to examine the status of PLC beta3 in the cardiac cell plasma membrane (sarcolemma, SL) in an experimental model of chronic diabetes. SL membrane was isolated from diabetic rat hearts at 8 weeks after a single i.v. injection of streptozotocin (65 mg/kg body weight). The total SL PLC was decreased in diabetes and was associated with a decrease in SL PLC beta3 activity, which immunofluorescence in frozen diabetic left ventricular tissue sections revealed to be due to a decrease in PLC beta3 protein abundance. In contrast, the SL abundance of Gqalpha was significantly increased during diabetes. These changes were associated with a loss of contractile function (+/- dP/dt). A 2-week insulin treatment of 6-week diabetic animals partially normalized all of these parameters. These findings suggest a defect in PLC beta3-mediated signaling processes may contribute to the cardiac dysfunction seen during diabetes.