Rationale: Reactive oxygen species generated during myocardial ischemia/reperfusion (I/R) potentiate myocyte death and cardiac dysfunction. Recently, we found a new, previously unappreciated role f...
Introduction: Proteostasis is the ability of the cell to balance the requirement of folding nascent proteins and degrading terminally misfolded ones in an efficient manner. A major site of proteost...
Fibroblasts in the heart respond to myocardial injury by infiltrating the affected area and differentiating into new cell types called myofibroblasts. These cells are characterized both by the induction of contractile proteins and the secretion of extracellular matrix proteins which form fibrotic scar tissue. Investigating the factors governing fibroblast activation is key to understanding how these cells function in the heart and may be key to future therapeutic strategies. Activating transcription factor 6 (ATF6), an effector of the endoplasmic reticulum unfolded protein response, plays critical roles in development, as well as in the differentiation of certain cell types, though it has not been studied in this regard in the heart. Our lab has demonstrated that ATF6 in cardiac myocytes is cardioprotective in vivo during heart disease. However, ATF6 has not been studied in cardiac fibroblasts and its effect on fibrosis in the heart is unknown. We hypothesized that ATF6 in fibroblasts is an important regulator of their function. Fibroblast activation markers including αSMA were increased in infarcted hearts with global ATF6 deletion. Additionally, hearts with pressure overload showed increased fibrosis staining in global ATF6-null mice relative to WT hearts. In isolated adult murine ventricular fibroblasts (AMVF), loss of ATF6 induced myofibroblast markers with and without the activation stimulus TGFβ. ATF6 loss of function also enhanced the effect of TGFβ on fibroblast contraction. These effects were associated with an increase in Smad phosphorylation, a crucial step in the TGFβ pathway. Interestingly, the effect of ATF6 loss of function in AMVF was erased when treated with a TGFβ receptor inhibitor. Additionally, when ATF6 was overexpressed or when endogenous ATF6 was chemically activated, myofibroblast markers were reduced and activation by TGFβ was blunted. ATF6 activation was associated with induction of several TGFβ/Smad pathway negative regulators including SMURF1, SMURF2, and PMEPA1, though none of these are known to be ATF6 target genes. These data suggest that ATF6 plays an important role in moderating fibroblast activation and this may contribute to previously reported roles for ATF6 in preserving cardiac function post-injury.
Introduction: Atrial natriuretic peptide (ANP) is a potent depressor hormone synthesized and stored in the heart in secretory granules of atrial myocytes as a biologically inactive precursor, pro-A...
The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.
The inbred mouse strain C57BL/6 is highly suitable and commonly used for the generation of transgenic mice as tools in dissecting biological processes and diseases. However, this parent line is available from a variety of sources with different sub‐strains. We examined male C57BL/6J mice from the Jackson Laboratory (n=29) and male C57BL/6NHsd from the Harlan Laboratories (n=36) in the setting of transverse aortic constriction (TAC), which is a common interventional procedure for studying pressure‐overload in hearts. Under anesthesia, the aortic arch between the innominate and the left carotid artery was constricted to 0.413 mm (27 ga needle) with a 7‐0 silk suture. Survival was monitored for 3 wks, and subsequently left ventricular function was assessed with a 1.4F Millar pressure transducer. Three wks after TAC, the survival rate was 50% for the C57BL/6NHsd and 79% survived for the C57BL/6J (Top Figure). A significant decline of 1058 mmHg/s (21%) in cardiac contractility (Bottom Figure) and 1228 mmHg/s (22%) in the rate of decay was found in C57BL/NHsd mice. Conclusion This study demonstrates phenotypic differences in viability and cardiac performance in response to TAC for these two sub‐strains. Therefore, it is imperative to carefully consider the appropriate line for the design of transgenic mice.
Pharmacologic activation of stress-responsive signaling pathways provides a promising approach for ameliorating imbalances in proteostasis associated with diverse diseases. However, this approach has not been employed in vivo . Here, using a mouse model of myocardial ischemia/reperfusion, we showed that selective pharmacologic activation of the ATF6 arm of the unfolded protein response (UPR) during reperfusion, a typical clinical intervention point after myocardial infarction, transcriptionally reprograms proteostasis, ameliorates damage and preserves heart function. These effects were lost upon cardiac myocyte-specific Atf6 deletion in the heart, demonstrating the critical role played by ATF6 in mediating pharmacologically activated proteostasis-based protection of the heart. Pharmacological activation of ATF6 was also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a first-in-class proteostasis-based therapeutic strategy for ameliorating ischemia/reperfusion damage, underscoring its unique translational potential for treating a wide range of pathologies caused by imbalanced proteostasis.
Introduction: Secreted and membrane proteins are synthesized and folded in the endoplasmic reticulum (ER). We previously showed that myocardial ischemia increases ER protein misfolding and activates the ER stress response, which is initially adaptive, fostering myocyte survival. However, if left unresolved, the ER stress response is maladaptive leading to myocyte death. ATF6 is an ER-transmembrane protein that senses misfolded proteins in the ER, and then becomes a transcription factor that induces ER stress response genes encoding ER proteins that adaptively restore ER protein folding. We previously showed that compared to non-transgenic mice, the hearts of ATF6 transgenic mice had better function and less damage upon ischemia/reperfusion (I/R); however, the mechanism of these protective effects is not known. Methods: Here, PCR gene array results showed that in cardiac myocytes, ATF6 induces genes encoding antioxidant proteins residing outside the ER, one of which is catalase. Thus, we explored the functions of ATF6-mediated catalase induction in the oxidative stress response during I/R in cardiac myocytes, in vitro and in vivo . Results: In vitro, overexpression of ATF6 in neonatal rat ventricular myocytes (NRVMs) induced protective ER stress response genes, while reducing reactive oxygen species (ROS), and necrotic cell death in cells treated with H 2 O 2 or simulated I/R (sI/R); ATF6 knockdown had the opposite effects. Catalase knockdown in NRVMs increased ROS, and necrotic cell death in cells treated with sI/R. ATF6 bound to putative ER stress response elements in the catalase gene and activated the catalase promoter. Mutation of these elements ablated ATF6 binding and ATF6-mediated catalase promoter activation. In vivo, compared to wild type, ATF6 knockout mouse hearts exhibited decreased catalase expression, reduced function and greater damage in response to myocardial infarction or I/R. Conclusions: This is the first demonstration in any cell type that ATF6 is critical for the adaptive oxidative stress response and the adaptive ER stress response, and describes a novel mechanism of ATF6-mediated protection of the heart from ischemic damage.
