Sex-specific phenotypes in the aging mouse heart and consequences for chronic fibrosis.

The incidence of diastolic dysfunction increases with age in both humans and mice. This is characterized by increased passive stiffness and slower relaxation of the left ventricle. The stiffness arises at least partially from progressively increased interstitial collagen deposition because of highly secretory fibroblasts. In the past, we demonstrated that AMPK activation via the drug 5-aminoimidazole-4-carboxamide riboside (AICAR) in middle-aged mice reduced adverse remodeling after myocardial infarction. Therefore, as an attempt to normalize the fibroblast phenotype, we used 21-mo-old male and female mice and treated them with AICAR (0.166 mg/g body wt) where each mouse was followed in a functional study over a 3-mo period. We found sex-related differences in extracellular matrix (ECM) composition as well as heart function indices at baseline, which were further accentuated by AICAR treatment. AICAR attenuated the age-related increase in left atrial volume (LAV, an indicator of diastolic dysfunction) in female but not in male hearts, which was associated with reduced collagen deposition in the old female heart, and reduced the transcription factor Gli1 expression in cardiac fibroblasts. We further demonstrated that collagen synthesis was dependent on Gli1, which is a target of AMPK-mediated degradation. By contrast, AICAR had a minor impact on cardiac fibroblasts in the old male heart because of blunted AMPK phosphorylation. Hence, it did not significantly improve old male heart function indices. In conclusion, we demonstrated that male and female hearts are phenotypically different, and sex-specific differences need to be considered when analyzing the response to pharmacological intervention.NEW & NOTEWORTHY The aging heart develops diastolic dysfunction because of increased collagen deposition. We attempted to reduce collagen expression in the old heart by activating AMPK using AICAR. An improvement of diastolic function and reduction of cardiac fibrosis was found only in the female heart and correlated with decreased procollagen expression and increased degradation of the transcription factor Gli1. Male hearts display blunted AICAR-dependent AMPK activation and therefore this treatment had no benefits for the male mice.

Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.

Coordinated changes in signaling pathways and gene expression in hearts subjected to prolonged stress maintain cardiac function. Loss of steroid receptor coactivator-2 (SRC-2) results in a reversal to the fetal gene program and disrupts the response to pressure overload, accompanied by prominent effects on metabolism and growth signaling, including increased AMPK activation. We proposed that early metabolic stress driven by AMPK activation induces contractile dysfunction in mice lacking SRC-2. We used 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK transiently before transverse aortic constriction (TAC) in wild-type and cardiomyocyte-specific SRC-2 knockout (CKO) animals. In contrast to AMPK activities during stress, in unstressed hearts, AICAR induced a mild activation of Akt signaling, and, in SRC-2-CKO mice, partially relieved an NAD+ deficiency and increased antioxidant signaling. These molecular changes translated to a mild hypertrophic response to TAC with decreased maladaptive remodeling, including markedly decreased fibrosis. Additionally, preactivation of AMPK in SRC-2-CKO mice was accompanied by a dramatic improvement in cardiac function compared with saline-treated SRC-2-CKO mice. Our results show that altered molecular signaling before stress onset has extended effects on sustained cardiac stress responses, and prestress modulation of transient growth and metabolism pathways may control those effects.-Nam, D. H., Kim, E., Benham, A., Park, H.-K., Soibam, B., Taffet, G. E., Kaelber, J. T., Suh, J. H., Taegtmeyer, H., Entman, M. L., Reineke, E. L. Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.

Steroid receptor coactivator-2 (SRC-2) coordinates cardiomyocyte paracrine signaling to promote pressure overload-induced angiogenesis.

Pressure overload-induced cardiac stress induces left ventricular hypertrophy driven by increased cardiomyocyte mass. The increased energetic demand and cardiomyocyte size during hypertrophy necessitate increased fuel and oxygen delivery and stimulate angiogenesis in the left ventricular wall. We have previously shown that the transcriptional regulator steroid receptor coactivator-2 (SRC-2) controls activation of several key cardiac transcription factors and that SRC-2 loss results in extensive cardiac transcriptional remodeling. Pressure overload in mice lacking SRC-2 induces an abrogated hypertrophic response and decreases sustained cardiac function, but the cardiomyocyte-specific effects of SRC-2 in these changes are unknown. Here, we report that cardiomyocyte-specific loss of SRC-2 (SRC-2 CKO) results in a blunted hypertrophy accompanied by a rapid, progressive decrease in cardiac function. We found that SRC-2 CKO mice exhibit markedly decreased left ventricular vasculature in response to transverse aortic constriction, corresponding to decreased expression of the angiogenic factor VEGF. Of note, SRC-2 knockdown in cardiomyocytes decreased VEGF expression and secretion to levels sufficient to blunt in vitro tube formation and proliferation of endothelial cells. During pressure overload, both hypertrophic and hypoxic signals can stimulate angiogenesis, both of which stimulated SRC-2 expression in vitro Furthermore, SRC-2 coactivated the transcription factors GATA-binding protein 4 (GATA-4) and hypoxia-inducible factor (HIF)-1α and -2α in response to angiotensin II and hypoxia, respectively, which drive VEGF expression. These results suggest that SRC-2 coordinates cardiomyocyte secretion of VEGF downstream of the two major angiogenic stimuli occurring during pressure overload bridging both hypertrophic and hypoxia-stimulated paracrine signaling.

AMP-activated protein kinase/myocardin-related transcription factor-A signaling regulates fibroblast activation and renal fibrosis.

Chronic kidney disease is a major cause of death, and renal fibrosis is a common pathway leading to the progression of this disease. Although activated fibroblasts are responsible for the production of the extracellular matrix and the development of renal fibrosis, the molecular mechanisms underlying fibroblast activation are not fully defined. Here we examined the functional role of AMP-activated protein kinase (AMPK) in the activation of fibroblasts and the development of renal fibrosis. AMPKα1 was induced in the kidney during the development of renal fibrosis. Mice with global or fibroblast-specific knockout of AMPKα1 exhibited fewer myofibroblasts, developed less fibrosis, and produced less extracellular matrix protein in the kidneys following unilateral ureteral obstruction or ischemia-reperfusion injury. Mechanistically, AMPKα1 directly phosphorylated cofilin leading to cytoskeleton remodeling and myocardin-related transcription factor-A nuclear translocation resulting in fibroblast activation and extracellular matrix protein production. Thus, AMPK may be a critical regulator of fibroblast activation through regulation of cytoskeleton dynamics and myocardin-related transcription factor-A nuclear translocation. Hence, AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

Aicar treatment reduces interstitial fibrosis in aging mice: Suppression of the inflammatory fibroblast.

In 2030, elderly people will represent 20% of the United States population. Even now, chronic cardiac diseases, especially heart failure with preserved systolic function (HFpEF), are the most expensive DRGs for Medicare. Progressive interstitial fibrosis in the aging heart is well recognized as an important component of HFpEF. Our recent studies suggested an important pathophysiologic role for reduced TGF-ß receptor 1 (TGFßR1) signaling in mesenchymal stem cells (MSCs) and their mesenchymal fibroblast progeny in the development of interstitial fibrosis. This report arises from our previous studies, which suggest that an inflammatory phenotype exists in these mesenchymal fibroblasts as a result of a reduced TGF-ß-Smad-dependent pathway but upregulated farnesyltransferase (FTase)-Ras-Erk signaling. In this report we provide evidence for a therapeutic approach that downregulates Erk activation through an adenosine monophosphate-activated kinase (AMPK) pathway. Aging C57BL/6J mice were treated with AICAR (an AMPK activator) for a 30-day period. This treatment suppressed excessive monocyte chemoattractant protein-1 (MCP-1) generation, which diminished leukocyte infiltration and in consequence suppressed the formation of macrophage-derived myeloid fibroblasts. Interestingly, the number of mesenchymal fibroblasts was also reduced. In addition, we observed changes in extracellular matrix (ECM) deposition, specifically that collagen type I and the alternatively spliced variant of fibronectin (EDA) expressions were reduced. These data suggest that the upregulation of AMPK activity is a potential therapeutic approach to fibrosis in the aging heart.

Dissecting the role of myeloid and mesenchymal fibroblasts in age-dependent cardiac fibrosis.

Aging is associated with increased cardiac interstitial fibrosis and diastolic dysfunction. Our previous study has shown that mesenchymal fibroblasts in the C57BL/6J (B6J) aging mouse heart acquire an inflammatory phenotype and produce higher levels of chemokines. Monocyte chemoattractant protein-1 (MCP-1) secreted by these aged fibroblasts promotes leukocyte uptake into the heart. Some of the monocytes that migrate into the heart polarize into M2a macrophages/myeloid fibroblasts. The number of activated mesenchymal fibroblasts also increases with age, and consequently, both sources of fibroblasts contribute to fibrosis. Here, we further investigate mechanisms by which inflammation influences activation of myeloid and mesenchymal fibroblasts and their collagen synthesis. We examined cardiac fibrosis and heart function in three aged mouse strains; we compared C57BL/6J (B6J) with two other strains that have reduced inflammation via different mechanisms. Aged C57BL/6N (B6N) hearts are protected from oxidative stress and fibroblasts derived from them do not develop an inflammatory phenotype. Likewise, these mice have preserved diastolic function. Aged MCP-1 null mice on the B6J background (MCP-1KO) are protected from elevated leukocyte infiltration; they develop moderate but reduced fibrosis and diastolic dysfunction. Based on these studies, we further delineated the role of resident versus monocyte-derived M2a macrophages in myeloid-dependent fibrosis and found that the number of monocyte-derived M2a (but not resident) macrophages correlates with age-related fibrosis and diastolic dysfunction. In conclusion, we have found that ROS and inflammatory mediators are necessary for activation of fibroblasts of both developmental origins, and prevention of either led to better functional outcomes.

Mesenchymal stem cell-derived inflammatory fibroblasts mediate interstitial fibrosis in the aging heart.

Pathologic fibrosis in the aging mouse heart is associated with dysregulated resident mesenchymal stem cells (MSC) arising from reduced stemness and aberrant differentiation into dysfunctional inflammatory fibroblasts. Fibroblasts derived from aging MSC secrete higher levels of 1) collagen type 1 (Col1) that directly contributes to fibrosis, 2) monocyte chemoattractant protein-1 (MCP-1) that attracts leukocytes from the blood and 3) interleukin-6 (IL-6) that facilitates transition of monocytes into myeloid fibroblasts. The transcriptional activation of these proteins is controlled via the farnesyltransferase (FTase)-Ras-Erk pathway. The intrinsic change in the MSC phenotype acquired by advanced age is specific for the heart since MSC originating from bone wall (BW-MSC) or fibroblasts derived from them were free of these defects. The potential therapeutic interventions other than clinically approved strategies based on findings presented in this review are discussed as well. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".

Mesenchymal stem cell-derived inflammatory fibroblasts promote monocyte transition into myeloid fibroblasts via an IL-6-dependent mechanism in the aging mouse heart.

Fibrosis in the old mouse heart arises partly as a result of aberrant mesenchymal fibroblast activation. We have previously shown that endogenous mesenchymal stem cells (MSCs) in the aged heart are markedly resistant to TGF-ß signaling. Fibroblasts originating from these MSCs retain their TGF-ß unresponsiveness and become inflammatory. In current studies, we found that these inflammatory fibroblasts secreted higher levels of IL-6 (3-fold increase, P < 0.05) when compared with fibroblasts derived from the young hearts. Elevated IL-6 levels in fibroblasts derived from old hearts arose from up-regulated expression of Ras protein-specific guanine nucleotide releasing factor 1 (RasGrf1), a Ras activator (5-fold, P < 0.01). Knockdown of RasGrf1 by gene silencing or pharmacologic inhibition of farnesyltransferase (FTase) or ERK caused reduction of IL-6 mRNA (more than 65%, P < 0.01) and decreased levels of secreted IL-6 (by 44%, P < 0.01). In vitro, IL-6 markedly increased monocyte chemoattractant protein-1-driven monocyte-to-myeloid fibroblast formation after transendothelial migration (TEM; 3-fold, P < 0.01). In conclusion, abnormal expression of RasGrf1 promoted production of IL-6 by mesenchymal fibroblasts in the old heart. Secreted IL-6 supported conversion of monocyte into myeloid fibroblasts. This process promotes fibrosis and contributes to the diastolic dysfunction in the aging heart.

Steroid receptor coactivator-2 is a dual regulator of cardiac transcription factor function.

We have previously demonstrated the potential role of steroid receptor coactivator-2 (SRC-2) as a co-regulator in the transcription of critical molecules modulating cardiac function and metabolism in normal and stressed hearts. The present study seeks to extend the previous information by demonstrating SRC-2 fulfills this role by serving as a critical coactivator for the transcription and activity of critical transcription factors known to control cardiac growth and metabolism as well as in their downstream signaling. This knowledge broadens our understanding of the mechanism by which SRC-2 acts in normal and stressed hearts and allows further investigation of the transcriptional modifications mediating different types and degrees of cardiac stress. Moreover, the genetic manipulation of SRC-2 in this study is specific for the heart and thereby eliminating potential indirect effects of SRC-2 deletion in other organs. We have shown that SRC-2 is critical to transcriptional control modulated by MEF2, GATA-4, and Tbx5, thereby enhancing gene expression associated with cardiac growth. Additionally, we describe SRC-2 as a novel regulator of PPARα expression, thus controlling critical steps in metabolic gene expression. We conclude that through regulation of cardiac transcription factor expression and activity, SRC-2 is a critical transcriptional regulator of genes important for cardiac growth, structure, and metabolism, three of the main pathways altered during the cardiac stress response.

CXCR6 plays a critical role in angiotensin II-induced renal injury and fibrosis.

OBJECTIVE: Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II-induced renal injury and fibrosis. APPROACH AND RESULTS: Wild-type and CXCR6-green fluorescent protein (GFP) knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg per minute after unilateral nephrectomy for ≤ 4 weeks. Wild-type and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between wild-type and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II-induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys after Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80(+) macrophages and CD3(+) T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment when compared with wild-type mice engrafted with CXCR6(+/+) bone marrow cells. CONCLUSIONS: Our results indicate that CXCR6 plays a pivotal role in the development of Ang II-induced renal injury and fibrosis through regulation of macrophage and T-cell infiltration and bone marrow-derived fibroblast accumulation.

Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts.

Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-ß), due to reduced expression of TGF-ß receptor I (TßRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-ß promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-ß signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TßRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ".

The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis.

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, and they proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. As chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly, the kidney of CXCR6 knockout mice accumulated fewer bone marrow-derived fibroblasts in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type (WT) mice. CXCR6 deficiency inhibited total collagen deposition and suppressed the expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, WT mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with WT mice engrafted with CXCR6(+/+) bone marrow cells. Transplant of WT bone marrow into CXCR6(-/-) recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may have important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

Aberrant differentiation of fibroblast progenitors contributes to fibrosis in the aged murine heart: role of elevated circulating insulin levels.

With age, the collagen content of the heart increases, leading to interstitial fibrosis. We have shown that CD44(pos) fibroblasts derived from aged murine hearts display reduced responsiveness to TGF-ß but, paradoxically, have increased collagen expression in vivo and in vitro. We postulated that this phenomenon was due to the defect in mesenchymal stem cell (MSC) differentiation in a setting of elevated circulating insulin levels and production that we observed in aging mice. We discovered that cultured fibroblasts derived from aged but not young cardiac MSCs of nonhematopoietic lineage displayed increased basal and insulin-induced (1 nM) collagen expression (2-fold), accompanied by increased farnesyltransferase (FTase) and Erk activities. In a quest for a possible mechanism, we found that a chronic pathophysiologic insulin concentration (1 nM) caused abnormal fibroblast differentiation of MSCs isolated from young hearts. Fibroblasts derived from these MSCs responded to insulin by elevating collagen expression as seen in untreated aged fibroblast cultures, suggesting a causal link between increased insulin levels and defective MSC responses. Here we report an insulin-dependent pathway that specifically targets collagen type I transcriptional activation leading to a unique mechanism of fibrosis that is TGF-ß and inflammation-independent in the aged heart.

Adiponectin promotes monocyte-to-fibroblast transition in renal fibrosis.

Bone marrow-derived fibroblasts may contribute substantially to the pathogenesis of renal fibrosis through the excessive production and deposition of extracellular matrix. However, the mechanisms underlying the accumulation and activation of these fibroblasts are not understood. Here, we used a mouse model of tubulointerstitial fibrosis to determine whether adiponectin, which is elevated in CKD and is associated with disease progression, regulates monocyte-to-fibroblast transition and fibroblast activation in injured kidneys. In wild-type mice, the expression of adiponectin and the number of bone marrow-derived fibroblasts in the kidney increased after renal obstruction. In contrast, the obstructed kidneys of adiponectin-knockout mice had fewer bone marrow-derived fibroblasts. Adiponectin deficiency also led to a reduction in the number of myofibroblasts, the expression of profibrotic chemokines and cytokines, and the number of procollagen-expressing M2 macrophages in injured kidneys. Consistent with these findings, adiponectin-deficiency reduced the expression of collagen I and fibronectin. Similar results were observed in wild-type and adiponectin-knockout mice after ischemia-reperfusion injury. In cultured bone marrow-derived monocytes, adiponectin stimulated the expression of α-smooth muscle actin (SMA) and extracellular matrix proteins and activated AMP-activated protein kinase (AMPK) in a time- and dose-dependent manner. Furthermore, specific activation of AMPK increased the expression of α-SMA and extracellular matrix proteins, while inhibition of AMPK attenuated these responses. Taken together, these findings identify adiponectin as a critical regulator of monocyte-to-fibroblast transition and renal fibrosis, suggesting that inhibition of adiponectin/AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

AICAR-dependent AMPK activation improves scar formation in the aged heart in a murine model of reperfused myocardial infarction.

We have demonstrated that scar formation after myocardial infarction (MI) is associated with an endogenous pool of CD44(pos)CD45(neg) multipotential mesenchymal stem cells (MSC). MSC differentiate into fibroblasts secreting collagen that forms a scar and mature into myofibroblasts that express alpha smooth muscle actin (α-SMA) that stabilizes the scar. In the aging mouse, cardiac repair after MI is associated with impaired differentiation of MSC; MSC derived from the aged hearts form dysfunctional fibroblasts that deposit less collagen in response to transforming growth factor beta-1 (TGF-ß1) and poorly mature into myofibroblasts. We found in vitro that the defect in myofibroblast maturation can be remedied by AICAR, which activates non-canonical TGF-ß signaling through AMP-activated protein kinase (AMPK). In the present study, we injected aged mice with AICAR and subjected them to 1h occlusion of the left anterior descending artery (LAD) and then reperfusion for up to 30days. AICAR-dependent AMPK signaling led to mobilization of an endogenous CD44(pos)CD45(neg) MSC and its differentiation towards fibroblasts and myofibroblasts in the infarct. This was accompanied by enhanced collagen deposition and collagen fiber maturation in the scar. The AICAR-treated group has demonstrated reduced adverse remodeling as indicated by improved apical end diastolic dimension but no changes in ejection fraction and cardiac output were observed. We concluded that these data indicate the novel, previously not described role of AMPK in the post-MI scar formation. These findings can potentially lead to a new therapeutic strategy for prevention of adverse remodeling in the aging heart.

TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis.

Angiotensin-II (Ang-II) is associated with many conditions involving heart failure and pathologic hypertrophy. Ang-II induces the synthesis of monocyte chemoattractant protein-1 that mediates the uptake of CD34(+)CD45(+) monocytic cells into the heart. These precursor cells differentiate into collagen-producing fibroblasts and are responsible for the Ang-II-induced development of non-adaptive cardiac fibrosis. In this study, we demonstrate that in vitro, using a human monocyte-to-fibroblast differentiation model, Ang-II required the presence of tumor necrosis factor-alpha (TNF) to induce fibroblast maturation from monocytes. In vivo, mice deficient in both TNF receptors did not develop cardiac fibrosis in response to 1week Ang-II infusion. We then subjected mice deficient in either TNF receptor 1 (TNFR1-KO) or TNF receptor 2 (TNFR2-KO) to continuous Ang-II infusion. Compared to wild-type, in TNFR1-KO, but not in TNFR2-KO hearts, collagen deposition was greatly attenuated, and markedly fewer CD34(+)CD45(+) cells were present. Quantitative RT-PCR demonstrated a striking reduction of key fibrosis-related, as well as inflammation-related mRNA expression in Ang-II-treated TNFR1-KO hearts. TNFR1-KO animals also developed less cardiac remodeling, cardiac hypertrophy, and hypertension compared to wild-type and TNFR2-KO in response to Ang-II. Our data suggest that TNF induced Ang-II-dependent cardiac fibrosis by signaling through TNFR1, which enhances the generation of monocytic fibroblast precursors in the heart.

Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction.

BACKGROUND: Delineating the role of microRNAs (miRNAs) in the posttranscriptional gene regulation offers new insights into how the heart adapts to pathological stress. We developed a knockout of miR-22 in mice and investigated its function in the heart. METHODS AND RESULTS: Here, we show that miR-22-deficient mice are impaired in inotropic and lusitropic response to acute stress by dobutamine. Furthermore, the absence of miR-22 sensitized mice to cardiac decompensation and left ventricular dilation after long-term stimulation by pressure overload. Calcium transient analysis revealed reduced sarcoplasmic reticulum Ca(2+) load in association with repressed sarcoplasmic reticulum Ca(2+) ATPase activity in mutant myocytes. Genetic ablation of miR-22 also led to a decrease in cardiac expression levels for Serca2a and muscle-restricted genes encoding proteins in the vicinity of the cardiac Z disk/titin cytoskeleton. These phenotypes were attributed in part to inappropriate repression of serum response factor activity in stressed hearts. Global analysis revealed increased expression of the transcriptional/translational repressor purine-rich element binding protein B, a highly conserved miR-22 target implicated in the negative control of muscle expression. CONCLUSION: These data indicate that miR-22 functions as an integrator of Ca(2+) homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.

Cardiovascular hemodynamics in mice with tumor necrosis factor receptor-associated factor 2 mediated cytoprotection in the heart.

Introduction: Many studies in mice have demonstrated that cardiac-specific innate immune signaling pathways can be reprogrammed to modulate inflammation in response to myocardial injury and improve outcomes. While the echocardiography standard parameters of left ventricular (LV) ejection fraction, fractional shortening, end-diastolic diameter, and others are used to assess cardiac function, their dependency on loading conditions somewhat limits their utility in completely reflecting the contractile function and global cardiovascular efficiency of the heart. A true measure of global cardiovascular efficiency should include the interaction between the ventricle and the aorta (ventricular-vascular coupling, VVC) as well as measures of aortic impedance and pulse wave velocity. Methods: We measured cardiac Doppler velocities, blood pressures, along with VVC, aortic impedance, and pulse wave velocity to evaluate global cardiac function in a mouse model of cardiac-restricted low levels of TRAF2 overexpression that conferred cytoprotection in the heart. Results: While previous studies reported that response to myocardial infarction and reperfusion was improved in the TRAF2 overexpressed mice, we found that TRAF2 mice had significantly lower cardiac systolic velocities and accelerations, diastolic atrial velocity, aortic pressures, rate-pressure product, LV contractility and relaxation, and stroke work when compared to littermate control mice. Also, we found significantly longer aortic ejection time, isovolumic contraction and relaxation times, and significantly higher mitral early/atrial ratio, myocardial performance index, and ventricular vascular coupling in the TRAF2 overexpression mice compared to their littermate controls. We found no significant differences in the aortic impedance and pulse wave velocity. Discussion: While the reported tolerance to ischemic insults in TRAF2 overexpression mice may suggest enhanced cardiac reserve, our results indicate diminished cardiac function in these mice.

Defective myofibroblast formation from mesenchymal stem cells in the aging murine heart rescue by activation of the AMPK pathway.

Aged mice in a murine model of myocardial infarction exhibit less effective myocardial repair. We hypothesized that the deficiency arises from altered lineage choice of endogenous mesenchymal stem cells (MSCs) and faulty maturation of myofibroblasts. Examination of cardiac MSCs revealed a substantial reduction in the pluripotency marker Nanog in cells from aged mice. In addition, the aged MSCs demonstrated a redirected lineage choice that favored adipocytic commitment over fibroblast or myofibroblast differentiation. Fibroblasts derived from aged MSCs demonstrated reduced expression of transforming growth factor-ß (TGF-ß) receptors I and II and diminished SMAD3 phosphorylation, associated with attenuated contractility and migration. Overexpression of constitutively active TGF-ß receptor I in aged cardiac fibroblasts ameliorated their defective motility but did not improve their contractility. Culturing of MSCs and fibroblasts with AICAR (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside) to activate adenosine monophosphate-activated kinase resulted in TGF-ß-dependent development of myofibroblasts with markedly enhanced contractility despite no reduction in adipocytic commitment or increased expression of TGF-ß receptors and SMAD3 phosphorylation. The data suggest an adenosine monophosphate-activated kinase-dependent gain of function as mediated by phosphorylation of TGF-ß-activated kinase 1 and p38 mitogen-activated protein kinase, which amplifies the response to TGF-ß1 via a non-canonical pathway, thus compensating for the reduced expression of TGF-ß receptors.

CXCL16 recruits bone marrow-derived fibroblast precursors in renal fibrosis.

Although fibroblasts are responsible for the production and deposition of extracellular matrix in renal fibrosis, their origin is controversial. Circulating fibroblast precursors may contribute to the pathogenesis of renal fibrosis, but the signaling mechanisms underlying the recruitment of bone marrow-derived fibroblast precursors into the kidney in response to injury are incompletely understood. Here, in the unilateral ureteral obstruction model of renal fibrosis, tubular epithelial cells upregulated the chemokine CXCL16 in obstructed kidneys, and circulating fibroblast precursors expressed the CXCL16 receptor, CXCR6. Compared with wild-type mice, CXCL16-knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors in obstructed kidneys. CXCL16-knockout mice also exhibited significantly fewer CD45-, collagen I-, and CXCR6-triple-positive fibroblast precursors in injured kidneys. Furthermore, targeted deletion of CXCL16 inhibited myofibroblast activation, reduced collagen deposition, and suppressed expression of collagen I and fibronectin. In conclusion, CXCL16 contributes to the pathogenesis of renal fibrosis by recruiting bone marrow-derived fibroblast precursors.