Indroneal Banerjee

Resident fibroblast lineages mediate pressure overload–induced cardiac fibrosis

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.

Dynamic Interactions between Myocytes, Fibroblasts, and Extracellular Matrix

Abstract:  Cardiac function is determined by the coordinated and dynamic interaction of several cell types together with components of the extracellular matrix (ECM). This interaction is regulated by mechanical, chemical, and electrical signals between the cellular and noncellular components of the heart. Recent studies using fluorescence‐activated cell sorting indicate that the number of myocytes remains relatively constant during development and disease, whereas the number of fibroblasts and other cell types can change dramatically. Cardiac fibroblasts appear to have different origins at different stages of development and fluctuate in response to a variety of physiological signals. Fibroblasts form a network of cells that are connected to each other via specific cadherins and connexins, to the ECM via integrins, and to myocytes by a variety of receptors, including connexins. Examples of the integration of signals include the role of angiotensin II (Ang II), which stimulates mechanical contraction of fibroblasts, as well as cytokine signaling. Cytokine signaling alters connexin and K+ channel activation, which in turn is regulated by Ang II, essentially forming a feedback loop. Quantitative changes in mechanical, chemical, and electrical signals that can alter the overall cardiac form and function will be discussed here.

Neonatal and adult cardiovascular pathophysiological remodeling and repair: developmental role of periostin.

The neonatal heart undergoes normal hypertrophy or compensation to complete development and adapt to increased systolic pressures. Hypertrophy and increased neonatal wall stiffness are associated with a doubling of the number of fibroblasts and de novo formation of collagen. Normal postnatal remodeling is completed within 3–4 weeks after birth but can be rekindled in adult life in response to environmental signals that lead to pathological hypertrophy, fibrosis, and heart failure. The signals that trigger fibroblast and collagen formation (fibrosis) as well as the origin and differentiation of the cardiac fibroblast lineage are not well understood. Using mice studies and a single‐cell engraftment model, we have shown that cardiac fibroblasts are derived from two extracardiac sources: the embryonic proepicardial organ and the recruitment of circulating bone marrow cells of hematopoietic stem cell origin. Periostin, a matricellular protein, is normally expressed in differentiating fibroblasts but its expression is elevated several fold in pathological remodeling and heart failure. Our hypothesis that periostin is profibrogenic (i.e., it promotes differentiation of progenitor mesenchymal cells into fibroblasts and their secretion and compaction of collagen) was tested using isolated and cultured embryonic, neonatal, and adult wild‐type and periostin‐null, nonmyocyte populations. Our findings indicate that abrogation of periostin by targeted gene deletion inhibits differentiation of nonmyocyte progenitor cells or permits misdirection into a cardiomyocyte lineage. However, if cultured with periostin or forced to express periostin, they became fibroblasts. Periostin plays a significant role in promoting fibrogenesis residual stress, and tensile testings indicated that periostin played an essential regulatory role in maintaining the biomechanical properties of the adult myocardium. These findings indicate that periostin is a profibrogenic matricellular protein that promotes collagen fibrogenesis, inhibits differentiation of progenitor cells into cardiomyocytes, and is essential for maintaining the biomechanical properties of the adult myocardium.

RhoA protects the mouse heart against ischemia/reperfusion injury.

The small GTPase RhoA serves as a nodal point for signaling through hormones and mechanical stretch. However, the role of RhoA signaling in cardiac pathophysiology is poorly understood. To address this issue, we generated mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (CA-RhoA mice) and demonstrated that they exhibited no overt cardiomyopathy. When challenged by in vivo or ex vivo ischemia/reperfusion (I/R), however, the CA-RhoA mice exhibited strikingly increased tolerance to injury, which was manifest as reduced myocardial lactate dehydrogenase (LDH) release and infarct size and improved contractile function. PKD was robustly activated in CA-RhoA hearts. The cardioprotection afforded by RhoA was reversed by PKD inhibition. The hypothesis that activated RhoA and PKD serve protective physiological functions during I/R was supported by several lines of evidence. In WT mice, both RhoA and PKD were rapidly activated during I/R, and blocking PKD augmented I/R injury. In addition, cardiac-specific RhoA-knockout mice showed reduced PKD activation after I/R and strikingly decreased tolerance to I/R injury, as shown by increased infarct size and LDH release. Collectively, our findings provide strong support for the concept that RhoA signaling in adult cardiomyocytes promotes survival. They also reveal unexpected roles for PKD as a downstream mediator of RhoA and in cardioprotection against I/R.

IL-6 loss causes ventricular dysfunction, fibrosis, reduced capillary density, and dramatically alters the cell populations of the developing and adult heart

Interleukin-6 (IL-6) is a pleiotropic cytokine responsible for many different processes including the regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs, including the heart. Recent studies have indicated that IL-6 is a critical component in the cell-cell communication between myocytes and cardiac fibroblasts. In this study, we examined the effects of IL-6 deficiency on the cardiac cell populations, cardiac function, and interactions between the cells of the heart, specifically cardiac fibroblasts and myocytes. To examine the effects of IL-6 loss on cardiac function, we used the IL-6(-/-) mouse. IL-6 deficiency caused severe cardiac dilatation, increased accumulation of interstitial collagen, and altered expression of the adhesion protein periostin. In addition, flow cytometric analyses demonstrated dramatic alterations in the cardiac cell populations of IL-6(-/-) mice compared with wild-type littermates. We observed a marked increase in the cardiac fibroblast population in IL-6(-/-) mice, whereas a concomitant decrease was observed in the other cardiac cell populations examined. Moreover, we observed increased cell proliferation and apoptosis in the developing IL-6(-/-) heart. Additionally, we observed a significant decrease in the capillary density of IL-6(-/-) hearts. To elucidate the role of IL-6 in the interactions between cardiac fibroblasts and myocytes, we performed in vitro studies and demonstrated that IL-6 deficiency attenuated the activation of the STAT3 pathway and VEGF production. Taken together, these data demonstrate that a loss of IL-6 causes cardiac dysfunction by shifting the cardiac cell populations, altering the extracellular matrix, and disrupting critical cell-cell interactions.

Thymosin Beta 4 Is Dispensable for Murine Cardiac Development and Function

Rationale: Thymosin beta 4 (T&bgr;4) is a 43–amino acid factor encoded by an X-linked gene. Recent studies have suggested that T&bgr;4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of t&bgr;4 has been reported to result in embryonic lethality at E14.5–16.5, with severe cardiac and angiogenic defects. However, this shRNA t&bgr;4-knockdown model did not completely abrogate T&bgr;4 expression. To completely ablate T&bgr;4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical. Objective: We examined the role of T&bgr;4 in developing and adult heart through global and cardiac specific t&bgr;4-knockout mouse models. Methods and Results: Global t&bgr;4-knockout mice were born at mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global t&bgr;4-knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue-specific t&bgr;4-deficient mice, generated by crossing t&bgr;4-floxed mice to Nkx2.5-Cre and &agr;MHC-Cre, were also found to have no phenotype. Conclusions: We conclude that T&bgr;4 is dispensable for embryonic viability, heart development, coronary vessel development, and adult myocardial function.

Targeted ablation of nesprin 1 and nesprin 2 from murine myocardium results in cardiomyopathy, altered nuclear morphology and inhibition of the biomechanical gene response.

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.

ALP/Enigma PDZ–LIM Domain Proteins in the Heart

Actinin-associated LIM protein (ALP) and Enigma are two subfamilies of Postsynaptic density 95, discs large and zonula occludens-1 (PDZ)-Lin-11, Isl1 and Mec-3 (LIM) domain containing proteins. ALP family members have one PDZ and one LIM domain, whereas Enigma proteins contain one PDZ and three LIM domains. Four ALP and three Enigma proteins have been identified in mammals, each having multiple splice variants and unique expression patterns. Functionally, these proteins bind through their PDZ domains to alpha-actinin and bind through their LIM domains or other internal protein interaction domains to other proteins, including signaling molecules. ALP and Enigma proteins have been implicated in cardiac and skeletal muscle structure, function and disease, neuronal function, bipolar disorder, tumor growth, platelet and epithelial cell motility and bone formation. This review will focus on recent advances in the biological roles of ALP/Enigma PDZ-LIM domain proteins in cardiac muscle and provide insights into mechanisms by which mutations in these proteins are related to human cardiac disease.

Excess protein O-GlcNAcylation and the progression of diabetic cardiomyopathy

We examined the role that enzymatic protein O-GlcNAcylation plays in the development of diabetic cardiomyopathy in a mouse model of Type 2 diabetes mellitus (DM2). Mice injected with low-dose streptozotocin and fed a high-fat diet developed mild hyperglycemia and obesity consistent with DM2. Studies were performed from 1 to 6 mo after initiating the DM2 protocol. After 1 mo, DM2 mice showed increased body weight, impaired fasting blood glucose, and hyperinsulinemia. Echocardiographic evaluation revealed left ventricular diastolic dysfunction by 2 mo and O-GlcNAcylation of several cardiac proteins and of nuclear transcription factor Sp1. By 4 mo, systolic dysfunction was observed and sarcoplasmic reticulum Ca(2+) ATPase expression decreased by 50%. Fibrosis was not observed at any timepoint in DM2 mice. Levels of the rate-limiting enzyme of the hexosamine biosynthetic pathway, glutamine:fructose-6-phosphate amidotransferase (GFAT) were increased as early as 2 mo. Fatty acids, which are elevated in DM2 mice, can possibly be linked to excessive protein O-GlcNAcylation levels, as cultured cardiac myocytes in normal glucose treated with oleic acid showed increased O-GlcNAcylation and GFAT levels. These data indicate that the early onset of diastolic dysfunction followed by the loss of systolic function, in the absence of cardiac hypertrophy or fibrosis, is associated with increased cardiac protein O-GlcNAcylation and increased O-GlcNAcylation levels of key calcium-handling proteins. A link between excessive protein O-GlcNAcylation and cardiac dysfunction is further supported by results showing that reducing O-GlcNAcylation by O-GlcNAcase overexpression improved cardiac function in the diabetic mouse. In addition, fatty acids play a role in stimulating excess O-GlcNAcylation. The nature and time course of changes observed in cardiac function suggest that protein O-GlcNAcylation plays a mechanistic role in the triggering of diabetic cardiomyopathy in DM2.

Pressure Overload Induces Early Morphological Changes in the Heart

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes.