Rationale: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes (T1D), accompanied by altered cardiac energetics, impaired mitochondrial function and oxidative stress. Previous studies indicate that T1D is associated with increased cardiac expression of Krüppel-like factor-5 (KLF5) and Peroxisome Proliferator Activated Receptor (PPAR)α that regulate cardiac lipid metabolism. Objective: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. Methods and Results: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and was higher compared with non-diabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacologic gain and loss of KLF5 function approaches and AAV-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 expression. This was accompanied by accumulation of cardiac ceramides. Pharmacologic or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation and improved cardiac function in diabetic mice. Conclusions: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation and causes DbCM.
Frequently, pharmacomechanisms are not fully elucidated. Therefore, drug use is linked to an elevated interindividual diversity of effects, whether therapeutic or adverse, and the role of biological sex has as yet unrecognized and underestimated consequences. A pharmacogenomic approach could contribute towards the development of an adapted therapy for each male and female patient, considering also other fundamental features, such as age and ethnicity. This would represent a crucial step towards precision medicine and could be translated into clinical routine. In the present review, we consider recent results from pharmacogenomics and the role of sex in studies that are relevant to cardiovascular therapy. We focus on genome-wide analyses, because they have obvious advantages compared with targeted single-candidate gene studies. For instance, genome-wide approaches do not necessarily depend on prior knowledge of precise molecular mechanisms of drug action. Such studies can lead to findings that can be classified into three categories: first, effects occurring in the pharmacokinetic properties of the drug, e.g. through metabolic and transporter differences; second, a pharmacodynamic or drug target-related effect; and last diverse adverse effects. We conclude that the interaction of sex with genetic determinants of drug response has barely been tested in large, unbiased, pharmacogenomic studies. We put forward the theory that, to contribute towards the realization of precision medicine, it will be necessary to incorporate sex into pharmacogenomics.
Cardiac hypertrophy manifests differently between men and women and there are pronounced sex differences in disease progression and outcome. To better understand remodeling and elucidate underlying mechanisms, most approaches have been limited to animal models or multicellular tissue samples. However, little is known about the role of biological sex in remodeling of human cardiomyocytes. We aimed at studying gene expression in isolated human cardiomyocytes under pressure overload hypothesizing significant sex differences. Cardiomyocytes were isolated from the left ventricular (LV) septum of aortic stenosis patients (n = 34; 50% men; 67.5 ± 9.6 years old) undergoing surgical aortic valve replacement. Patients underwent 2D‐guided M‐mode transthoracic echocardiography during the week before surgery and 20.06 ± 9.7 days after surgery. We measured with qRT‐PCR structural genes, as they are involved in cardiac hypertrophy and remodeling, thereby influencing cardiac function. The study was conducted in conformance with the Basic Principles of the Declaration of Helsinki. There was no significant difference between men and women in age, body mass index, systolic and diastolic blood pressure, co‐morbidities and medication. Preoperative posterior wall thickness (P = 0.02), LV end diastolic diameter (P = 0.007) and mass index (P = 0.03) were higher in men than women. At similar levels of aortic valve area index between the sexes, preoperative ejection fraction (EF) was significantly lower in men than women (P = 0.01). Postoperative EF levels improved in men reaching those of women. Male cardiomyocytes had significantly higher levels of ACTC1 (P = 0.03), CTGF (P = 0.03), GATA4 (P = 0.03), GJA1 (P = 0.03), MYH6 (P = 0.02), MYL4 (P = 0.03), NFKB1 (P = 0.01), NPPA (P = 0.03) and NPPB (P = 0.05) than female cardiomyocytes. Next, we asked whether there is an association between gene expression and postoperative EF. The expression of GATA4 (r = −0.77, P = 0.01), GJA1 (r = −0.69, P = 0.03), MYH6 (r = −0.69, P = 0.04), MYH7 (r = −0.82, P = 0.008), MYL4 (r = −0.71, P = 0.03), NPPA (r = −0.9, P = 0.001) and NPPB (r = −0.79, P = 0.01) was negatively correlated with postoperative EF in men only. In conclusion, gene expression is regulated in a sex‐specific manner in isolated cardiomyocytes of aortic stenosis patients. Compared with women, men had higher levels of maladaptive remodeling factors, which may be part of the molecular mechanisms underlying the observed sex differences in cardiac function, i.e. EF. Given the male‐specific changes in EF postoperatively, cardiomyocyte‐specific gene regulation at the time of surgery might inform disease progression.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introduction: Cardiomyopathy in type 1 diabetes (T1D) is accompanied by impaired mitochondrial function, oxidative stress and lipotoxicity. We showed that cardiomyocyte (CM) Krüppel-like factor 5 (KLF5) is increased in streptozotocin-induced T1D and induces Peroxisome Proliferator Activated Receptor (PPAR)α in mice. Hypothesis: KLF5 upregulation by FOXO1 induces diabetic cardiomyopathy (DbCM). Methods and Results: Analyses in CM from diabetic patients showed higher KLF5 mRNA levels compared to non-diabetic individuals. In vitro mechanistic and in vivo analyses in αMHC- Foxo1 -/- mice revealed that FOXO1 stimulates KLF5 expression via direct promoter binding. Genetic inhibition of CM FOXO1 alleviated DbCM. Additionally, AAV-mediated CM-specific KLF5 overexpression in C57Bl/6 (WT) mice induced cardiac dysfunction. Mice with CM-specific KLF5 constitutive expression (αMHC-rtTA- Klf5 ), which we generated, recapitulated cardiomyopathy without T1D. Moreover, Pparα -/- mice with T1D, had higher CM-KLF5 levels and developed DbCM, suggesting that KLF5-driven DbCM is PPARα-independent. Additionally, CM-KLF5 induced oxidative stress through increased NADPH oxidase (NOX)4 expression and lower mitochondria abundance. Conversely, KLF5 inhibition prevented NOX4 upregulation and superoxide formation. Furthermore, CM-KLF5 promoted NOX4 expression via direct promoter binding. Antioxidant treatment in diabetic WT and αMHC-rtTA- Klf5 mice alleviated cardiac dysfunction partially, suggesting other pathways that contribute in KLF5-induced DbCM. For that, we performed cardiac lipidome analysis where we found clustering of αMHC-rtTA- Klf5 with diabetic WT mice. Of note, KLF5 inhibition in diabetic mice resulted in similar lipidome with non-diabetic WT mice. Individual lipid species analysis showed increased ceramide accumulation in diabetic WT and αMHC-rtTA- Klf5 mice that was reversed upon KLF5 inhibition. Thus, CM-KLF5 activation correlates with cardiac ceramide accumulation, that has been associated with cardiac lipotoxicity. Conclusions: In conclusion, T1D stimulates FOXO1, which induces CM-KLF5 expression that leads to oxidative stress and DbCM in a non-PPARα-dependent manner, as well as to cardiac ceramide accumulation.
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