Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/guidelines-for-evaluating-myocardial-cell-death/ .

A three-dimensional (3-D) cell culture system that allows control of both substrate stiffness and integrin binding density was created and characterized. This system consisted of two self-assembling peptide (SAP) sequences that were mixed in different ratios to achieve the desired gel stiffness and adhesiveness. The specific peptides used were KFE ((acetyl)-FKFEFKFE-CONH2), which has previously been reported not to support cell adhesion or MVN formation, and KFE-RGD ((acetyl)-GRGDSP-GG-FKFEFKFE-CONH2), which is a similar sequence that incorporates the RGD integrin binding site. Storage modulus for these gels ranged from ~60 to 6000 Pa, depending on their composition and concentration. Atomic force microscopy revealed ECM-like fiber microarchitecture of gels consisting of both pure KFE and pure KFE-RGD as well as mixtures of the two peptides. This system was used to study the contributions of both matrix stiffness and adhesiveness on microvascular network (MVN) formation of endothelial cells and the morphology of human mesenchymal stem cells (hMSC). When endothelial cells were encapsulated within 3-D gel matrices without binding sites, little cell elongation and no network formation occurred, regardless of the stiffness. In contrast, matrices containing the RGD binding site facilitated robust MVN formation, and the extent of this MVN formation was inversely proportional to matrix stiffness. Compared with a matrix of the same stiffness with no binding sites, a matrix containing RGD-functionalized peptides resulted in a ~2.5-fold increase in the average length of network structure, which was used as a quantitative measure of MVN formation. Matrices with hMSC facilitated an increased number and length of cellular projections at higher stiffness when RGD was present, but induced a round morphology at every stiffness when RGD was absent. Taken together, these results demonstrate the ability to control both substrate stiffness and binding site density within 3-D cell-populated gels and reveal an important role for both stiffness and adhesion on cellular behavior that is cell-type specific.

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Sensory feedback is essential for acquiring and maintaining complex motor behaviors, including birdsong. In zebra finches, auditory feedback reaches the song control circuits primarily through the nucleus interfacialis nidopalii (Nif), which provides excitatory input to HVC (proper name)-a premotor region essential for the production of learned vocalizations. Despite being one of the major inputs to the song control pathway, the role of Nif in generating vocalizations is not well understood. To address this, we transiently inactivated Nif in late juvenile zebra finches. Upon Nif inactivation (in both hemispheres or on one side only), birds went from singing stereotyped zebra finch song to uttering highly variable and unstructured vocalizations resembling sub-song, an early juvenile song form driven by a basal ganglia circuit. Simultaneously inactivating Nif and LMAN (lateral magnocellular nucleus of the anterior nidopallium), the output nucleus of a basal ganglia circuit, inhibited song production altogether. These results suggest that Nif is required for generating the premotor drive for song. Permanent Nif lesions, in contrast, have only transient effects on vocal production, with song recovering within a day. The sensorimotor nucleus Nif thus produces a premotor drive to the motor pathway that is acutely required for generating learned vocalizations, but once permanently removed, the song system can compensate for its absence. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1213-1225, 2016.