Diabetic cardiomyopathy (DbCM), which consists of cardiac hypertrophy and failure in the absence of traditional risk factors, is a major contributor to increased heart failure risk in type 2 diabetes patients. In rodent models of DbCM, cardiac hypertrophy and dysfunction have been shown to depend upon saturated fatty acid (SFA) oversupply and de novo sphingolipid synthesis. However, it is not known whether these effects are mediated by bulk SFAs and sphingolipids or by individual lipid species. In this report, we demonstrate that a diet high in SFA induced cardiac hypertrophy, left ventricular systolic and diastolic dysfunction, and autophagy in mice. Furthermore, treatment with the SFA myristate, but not palmitate, induced hypertrophy and autophagy in adult primary cardiomyocytes. De novo sphingolipid synthesis was required for induction of all pathological features observed both in vitro and in vivo, and autophagy was required for induction of hypertrophy in vitro. Finally, we implicated a specific ceramide N-acyl chain length in this process and demonstrated a requirement for (dihydro)ceramide synthase 5 in cardiomyocyte autophagy and myristate-mediated hypertrophy. Thus, this report reveals a requirement for a specific sphingolipid metabolic route and dietary SFAs in the molecular pathogenesis of lipotoxic cardiomyopathy and hypertrophy. IntroductionObesity and diabetes present two of the most important health challenges facing the Western world at this time. Patients suffering from type 2 diabetes (T2D) are subject to a number of major health risks, including a greatly increased risk of heart failure (1). This is due in part to the development of diabetic cardiomyopathy (DbCM), which occurs independently of other traditional risk factors (2). DbCM promotes cardiac remodeling and impairs cardiac function (1). Importantly, individuals with T2D and the metabolic syndrome present with dyslipidemia, and recent studies have suggested that DbCM may occur as a result of lipid overload and subsequent lipotoxic events (ref. 2, reviewed in ref. 3). In particular, oversupply of saturated fatty acids (SFAs) has been implicated in this process.Previous studies of lipotoxic DbCM in rodents have relied on several important transgenic models. The B6.Cg-Lep ob /J (ob/ob) and B6.BKS(D)-Lepr db /J (db/db) mouse models, which lack the genes encoding leptin and the leptin receptor, respectively, are both very popular models that develop obesity and a DbCM-like cardiac phenotype (reviewed in ref. 4). Other less common models, including the Atgl-knockout mouse and the LpL GPI transgenic mouse, also induce lipotoxic cardiomyopathy by perturbing cardiac lipid uptake, handling, or metabolism (reviewed in ref. 4). While these transgenic models robustly induce lipid overload in cardiomyocytes, the very disruptions that produce lipotoxic DbCM also drastically alter patterns of lipid uptake and handling in a nonphysiologic way. In contrast, wild-type mice fed standard lard-based high-fat diets (LBD) failed to develop a DbCM-like phenotyp...

Increased myocardial extracellular matrix collagen represents an important structural milestone during the development of left ventricular (LV) pressure overload (PO); however, the proteolytic pathways that contribute to this process are not fully understood. This study tested the hypothesis that membrane type 1-matrix metalloproteinase (MT1-MMP) is directly induced at the transcriptional level in vivo during PO and is related to changes in LV collagen content. PO was induced in vivo by transverse aortic constriction in transgenic mice containing the full length human MT1-MMP promoter region ligated to luciferase (MT1-MMP Prom mice). MT1-MMP promoter activation (luciferase expression), expression, and activity; collagen volume fraction (CVF); and left atrial dimension were measured at 1 (n = 8), 2 (n = 12), and 4 (n = 17) wk following PO. Non-PO mice (n = 10) served as controls. Luciferase expression increased by fivefold at 1 wk, fell at 2 wk, and increased again by ninefold at 4 wk of PO (P < 0.05). MT1-MMP expression and activity increased at 1 wk, fell at 2 wk, and increased again at 4 wk after PO. CVF increased at 1 wk, remained unchanged at 2 wk, and increased by threefold at 4 wk of PO (P < 0.05). There was a strong positive correlation between CVF and MT1-MMP activity (r = 0.80, P < 0.05). Left atrial dimension remained unchanged at 1 and 2 wk but increased by 25% at 4 wk of PO. When a mechanical load was applied in vitro to LV papillary muscles isolated from MT1-MMP Prom mice, increased load caused MT1-MMP promoter activation to increase by twofold and MT1-MMP expression to increase by fivefold (P < 0.05). These findings challenge the canonical belief that PO suppresses overall matrix proteolytic activity, but rather supports the concept that certain proteases, such as MT1-MMP, play a pivotal role in PO-induced matrix remodeling and fibrosis.

Background MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by Class I and IIb HDACs during hypertrophic remodeling. Methods and Results Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload (PO) hypertrophy. Mice were treated with Class I and IIb HDAC inhibitor via drinking water for 2 and 4 weeks post-TAC. miRNA expression was determined by real time PCR. Echocardiography was performed at baseline and post-TAC endpoints for structural and functional assessment. Chromatin immunoprecipitation (ChIP) was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7 and 0.5 fold at 2 weeks and 4 weeks post-TAC respectively as compared to vehicle-control (P < 0.05). HDAC inhibition prevented this significant decrease 2 weeks post-TAC and maintained miR-133a expression near vehicle-control levels, which coincided with 1) a decrease in connective tissue growth factor (CTGF) expression, 2) a reduction in cardiac fibrosis and left atrium diameter (marker of end-diastolic pressure) suggesting an improvement in diastolic function. ChIP analysis revealed that HDAC1 and HDAC2 are present on the miR-133a enhancer regions. Conclusions The results reveal that HDACs play a role in the regulation of PO-induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in PO-induced cardiac fibrosis.

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Background: The expression level of several microRNAs (miRNAs) is affected by pressure overload hypertrophy and has an impact on cardiac function. Histone deacetylases (HDACs) regulate the transcription of many genes whose expression is altered in hypertrophy. Therefore, we hypothesize that acetylation regulates the expression of some miRs during cardiac hypertrophy . Methods: Transverse aortic constriction (TAC) was performed to initiate cardiac hypertrophy induced by pressure overload. Mice were treated with the Class I and IIb HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) in the drinking water post-TAC for 2 and 4 weeks. miRNAs expression normalized to RNU6B was determined by qRT-PCR. Echocardiography was performed at baseline and post-TAC endpoints to assess physiological parameters. Chromatin immunoprecipitation (ChIP) was used to identify HDACs and transcription factors associated with miR-133a promoter. Results: Based on previous studies, we selected 8 miRNAs that play major roles in hypertrophy and assessed the effect of SAHA treatment on their expression level. The expression of one of the miRNAs, miR-133a was significantly (P < 0.05) downregulated at both 2 weeks (1887 ±105, n=8) and 4 weeks post-TAC (1651 ±103, n=9) compared to control group (2972 ±334, n=11). SAHA treatment significantly de-repressed miR-133a expression 2 weeks post-TAC (3317 ±560, n=12) and to a lesser extent after 4 weeks post-TAC (2391 ±455, n=10). As expected, left atrium (LA) diameter, a marker of diastolic pressure, increased 2 weeks post-TAC, while SAHA treatment significantly reduced LA diameter. miR-133a targets connective tissue growth factor (CTGF) and collagen 1a1. Consistent with SAHA-mediated miR-133a de-repression, CTGF expression and collagen volume fraction were decreased compared to TAC alone. Importantly, ChIP analysis revealed that HDAC2 was present on the miR-133a promoter. Conclusion: We show that HDAC2, a Class I HDAC, plays a role in the regulation of miR-133a expression in cardiac hypertrophy. HDACs and miRNAs are key regulators of the events mediating cardiac pathology. Our work demonstrates that HDAC inhibition may be an attractive therapeutic strategy for the regulation of some miRNAs in heart disease.