Rationale:: Endoplasmic reticulum (ER) stress causes the accumulation of misfolded proteins in the ER, activating the transcription factor, ATF6 (activating transcription factor 6 alpha), which induces ER stress response genes. Myocardial ischemia induces the ER stress response; however, neither the function of this response nor whether it is mediated by ATF6 is known. Objective:: Here, we examined the effects of blocking the ATF6-mediated ER stress response on ischemia/reperfusion (I/R) in cardiac myocytes and mouse hearts. Methods and Results:: Knockdown of ATF6 in cardiac myocytes subjected to I/R increased reactive oxygen species and necrotic cell death, both of which were mitigated by ATF6 overexpression. Under nonstressed conditions, wild-type and ATF6 knockout mouse hearts were similar. However, compared with wild-type, ATF6 knockout hearts showed increased damage and decreased function after I/R. Mechanistically, gene array analysis showed that ATF6, which is known to induce genes encoding ER proteins that augment ER protein folding, induced numerous oxidative stress response genes not previously known to be ATF6-inducible. Many of the proteins encoded by the ATF6-induced oxidative stress genes identified here reside outside the ER, including catalase, which is known to decrease damaging reactive oxygen species in the heart. Catalase was induced by the canonical ER stressor, tunicamycin, and by I/R in cardiac myocytes from wild-type but not in cardiac myocytes from ATF6 knockout mice. ER stress response elements were identified in the catalase gene and were shown to bind ATF6 in cardiac myocytes, which increased catalase promoter activity. Overexpression of catalase, in vivo, restored ATF6 knockout mouse heart function to wild-type levels in a mouse model of I/R, as did adeno-associated virus 9–mediated ATF6 overexpression. Conclusions:: ATF6 serves an important role as a previously unappreciated link between the ER stress and oxidative stress gene programs, supporting a novel mechanism by which ATF6 decreases myocardial I/R damage.

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 we show, using a mouse model of myocardial ischemia/reperfusion, 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 is also protective in renal and cerebral ischemia/reperfusion models, demonstrating its widespread utility. Thus, pharmacologic activation of ATF6 represents a 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.

Activating transcription factor-6 α (ATF6) is one of the three main sensors and effectors of the endoplasmic reticulum (ER) stress response and, as such, it is critical for protecting the heart and other tissues from a variety of environmental insults and disease states. In the heart, ATF6 has been shown to protect cardiac myocytes. However, its roles in other cell types in the heart are unknown. Here we show that ATF6 decreases the activation of cardiac fibroblasts in response to the cytokine, transforming growth factor β (TGFβ), which can induce fibroblast trans-differentiation into a myofibroblast phenotype through signaling via the TGFβ–Smad pathway. ATF6 activation suppressed fibroblast contraction and the induction of α smooth muscle actin (αSMA). Conversely, fibroblasts were hyperactivated when ATF6 was silenced or deleted. ATF6 thus represents a novel inhibitor of the TGFβ–Smad axis of cardiac fibroblast activation.

The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.

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.