Despite the considerable work that has been performed to tease apart the mechanisms of lipotoxic DbCM, to date, only general information has been published about the specific lipid mediators governing this process. In particular, although a few studies have implicated ceramides as a lipid class required for induction of cardiac lipotoxicity, the particular species involved have yet to be determined (23
). Additionally, the practical relevance of lipotoxicity to DbCM remained to be demonstrated, as all effective models of DbCM, to date, relied on genetic manipulations that disrupted cardiac lipid handling in a manner with no clinical parallels (ref. 2
, reviewed in refs. 3
The present study addresses both of these concerns. Here, we present a nontransgenic model of diet-induced obesity that robustly produced DbCM-like cardiac hypertrophy and functional impairment. Our findings revealed that an obesogenic diet based on milk fat, rather than lard, induced cardiac dysfunction, both gross and cellular hypertrophy, and increased autophagy in hearts of nontransgenic mice (Figures and , and Tables and ). Furthermore, all of these outcomes were shown to be sphingolipid dependent. This diet, which was highly enriched in myristate (C14:0), also specifically potentiated production of the myristate-containing ceramide species C14-ceramide (Figure ). These findings provided a compelling argument for further in vitro mechanistic work in isolated adult cardiomyocytes, where we were able to identify a specific pathway through which oversupply of an individual saturated fat induced cardiac hypertrophy. In isolated cells, myristate, but not palmitate (C16:0), was found to induce sphingolipid-dependent cardiomyocyte hypertrophy (Figure ). Furthermore, myristate treatment potentiated C14-ceramide production, as was seen in the animal model. Subsequent to these observations, we identified a specific CerS isoform (CerS5) required for induction of hypertrophy by lipid overload (Figure ).
This work also provided basic insight into the link between autophagy and lipotoxic cardiomyocyte hypertrophy. While numerous reports have demonstrated an increase in autophagy in other hypertrophic cardiac conditions, very few have examined cardiac autophagy in the context of T2D, and none have determined what effect lipid overload has on this pathway. Additionally, some controversy has surrounded the role for autophagy in the hypertrophic heart (31
). The debate centers on whether autophagy promotes or protects against cardiac hypertrophy in these systems. One major hypothesis unifies these 2 points of view, suggesting that autophagy is a “Goldilocks” phenomenon: too little is pathogenic; too much is pathogenic (50
). In an isolated cardiomyocyte system, we demonstrated that oversupply of myristate, but not palmitate, promoted an increase in autophagy (Figures and ). Furthermore, we directly tested whether inhibition of autophagy would promote hypertrophy or prevent it. In fact, we found that fatty acid–induced hypertrophy was completely prevented by knockdown of LC3B or 3-methyladenine treatment, suggesting a pathogenic role for autophagy in cardiac lipid overload (Figure and Supplemental Figure 8). Finally, we linked the induction of autophagy by myristate to the same CerS isoform implicated in the hypertrophic response (Figures and ).
Based on these findings, we propose a model in which a diet rich in myristate, which is a major constituent of milk fat–containing products such as butter and ice cream, induces cardiac hypertrophy through its effects on ceramide synthesis and composition. Namely, dietary myristate oversupply promotes ceramide synthesis via CerS5 and, consequently, increased autophagy. This increase in autophagy subsequently promotes hypertrophy through a mechanism that has yet to be identified.
Two major routes could potentially link autophagy and hypertrophy. The first mechanism would be mediated by conventional signaling pathways. While investigations remain ongoing, several potential connections have emerged through bioinformatic analyses. In particular, analysis of protein-protein interaction pathways has revealed links among 3 proteins of the autophagy pathway (i.e., Atg7, Atg12, and RB1CC1/FIP200) and the hypertrophy protein GSK3β via several routes, including FOXO1 and SIRT1 (S.B. Russo, unpublished data). Indeed, literature has already implicated FOXO1 and SIRT1 in regulation of both autophagy and hypertrophy in cardiomyocytes (51
). Thus, it appears likely that autophagy and hypertrophy pathways may be subject to common regulators and could possibly engage in crosstalk.
The authors also hypothesize a second, less obvious mechanism by which upregulation of autophagy may be required for induction of hypertrophy. To develop the hypertrophic phenotype, cardiomyocytes undergo substantial subcellular remodeling of numerous components, including the mitochondria, sarcolemmal membrane, sarcoplasmic reticulum, and sarcomeres (55
). Autophagy is a key process of cellular remodeling, and in particular, remodeling and recycling of the endoplasmic/sarcoplasmic reticulum and mitochondria (56
). On this basis, the authors speculate that, by altering the intracellular landscape via organelle turnover and by recycling intracellular components, induction of autophagy potentiates the organized remodeling prompted by prohypertrophic stimuli.
With regard to the mechanism linking C14
-ceramide levels to autophagy, the authors of this study hypothesize that, in addition to upstream signaling roles, the effect of increased C14
-ceramide may mediate membrane properties of the autophagosome and, thereby, the activity of membrane-resident proteins involved in autophagy. This notion is based on data from the literature that indicate that sphingolipid N
-acyl chain lengths alter the biophysical properties of membranes, including curvature, fluidity, stability, and permeability (58
). In particular, it has been shown that genetic ablation of CerS2
in mice promoted membrane fusion and budding (66
). This was mechanistically attributed to alterations in lipid packing and membrane curvature, which occurred due to a shift toward shorter, unsaturated ceramides and a total ablation of C22
- to C24
-ceramide production. Furthermore, it has been shown that the activities of membrane-resident proteins are modulated by membrane biophysical properties and are sensitive to membrane lipid composition (67
). In findings particularly relevant to this study, it was shown that membrane lipid composition and curvature were essential for targeting of Barkor/Atg14 to the budding autophagosome (70
). Additionally, it has been shown that the proautophagic activity of BECN1 is mediated by its exit from sphingolipid-enriched membrane microdomains (so-called “lipid rafts”) in the context of traumatic brain injury (71
). Thus, it seems plausible that increases in C14
-ceramide may induce autophagy by promoting membrane budding and fusion and by altering localization or activity of membrane-resident proteins, including BECN1.
The diet model used in this study also represents an important technical advance in the study of lipotoxicity in DbCM. Specifically, previous studies of lipotoxic DbCM in mice have relied upon transgenic mouse models, including the ob/ob
strains (reviewed in refs. 3
). While these models closely mimic the DbCM phenotype, they do so by grossly perturbing cardiac metabolism and lipid handling. Thus, by their very nature, these studies confound any meaningful inference about roles for specific fatty acids and sphingolipid species in the pathophysiology of genotypically normal diabetic myocardium. Further complicating this effort, previously investigated diet-induced obesity models, which utilize high-fat feeding in wild-type mice, poorly reproduced the cardiac parameters of DbCM, including hypertrophy. This may be because the fatty acid profile of lard, which is the fat source for these diets, contains close to 60% combined oleic and linoleic acids, which have been shown to protect against lipotoxicity and might be expected to attenuate lipotoxic outcomes (11
In contrast, in the present study, feeding mice a diet based on milk fat, which is high in saturated fat and low in protective UFAs, robustly induced a DbCM phenotype (Tables and ). Furthermore, because this outcome was achieved in wild-type mice, meaningful conclusions could be drawn about the lipid species involved in this process. Thus, the present model represents a new and powerful tool to dissect the mechanisms underlying lipotoxic cardiomyopathy in a physiologically relevant context.
In conclusion, we present here what we believe is a new diet-based model of DbCM. This new model led us to define a specific role for C14-ceramide and CerS5 in promoting cardiac autophagy and subsequent hypertrophy of cardiomyocytes. Thus, this study implicates a specific ceramide species and CerS isoform in autophagy, hypertrophy, and cardiomyopathy resulting from lipid overload. Additionally, it provides a specific biochemical chain leading from dietary saturated fat to sphingolipid-mediated cardiac hypertrophy. Future studies aim to address the role for CerS5 in diet-induced obesity and insulin resistance in vivo as well as to identify the specific mechanisms by which myristate and C14-ceramide promote autophagy and hypertrophy.