Qinglin Yang

Cardiomyocyte-restricted peroxisome proliferator-activated receptor-|[delta]| deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy

Fatty acid oxidation (FAO) is a primary energy source for meeting the hearts energy requirements. Peroxisome proliferator-activated receptor-δ (PPAR-δ) may have important roles in FAO. But it remains unclear whether PPAR-δ is required for maintaining basal myocardial FAO. We show that cre-loxP-mediated cardiomyocyte-restricted deletion of PPAR-δ in mice downregulates constitutive expression of key FAO genes and decreases basal myocardial FAO. These mice have cardiac dysfunction, progressive myocardial lipid accumulation, cardiac hypertrophy and congestive heart failure with reduced survival. Thus, chronic myocardial PPAR-δ deficiency leads to lipotoxic cardiomyopathy. Together, our data show that PPAR-δ is a crucial determinant of constitutive myocardial FAO and is necessary to maintain energy balance and normal cardiac function. We suggest that PPAR-δ is a potential therapeutic target in treating lipotoxic cardiomyopathy and other heart diseases.

A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy can be caused by mutations in genes encoding sarcomeric proteins, including the cardiac isoform of myosin binding protein C (MyBP-C), and multiple mutations which cause truncated forms of the protein to be made are linked to the disease. We have created transgenic mice in which varying amounts of a mutated MyBP-C, lacking the myosin and titin binding domains, are expressed in the heart. The transgenically encoded, truncated protein is stable but is not incorporated efficiently into the sarcomere. The transgenic muscle fibers showed a leftward shift in the pCa2+-force curve and, importantly, their power output was reduced. Additionally, expression of the mutant protein leads to decreased levels of endogenous MyBP-C, resulting in a striking pattern of sarcomere disorganization and dysgenesis.

Inactivation of focal adhesion kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in mice

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in integrin signaling pathways. Although cardiovascular defects were observed in FAK total KO mice, the embryonic lethality prevented investigation of FAK function in the hearts of adult animals. To circumvent these problems, we created mice in which FAK is selectively inactivated in cardiomyocytes (CFKO mice). We found that CFKO mice develop eccentric cardiac hypertrophy (normal LV wall thickness and increased left chamber dimension) upon stimulation with angiotensin II or pressure overload by transverse aortic constriction as measured by echocardiography. We also found increased heart/body weight ratios, elevated markers of cardiac hypertrophy, multifocal interstitial fibrosis, and increased collagen I and VI expression in CFKO mice compared with control littermates. Spontaneous cardiac chamber dilation and increased expression of hypertrophy markers were found in the older CFKO mice. Analysis of cardiomyocytes isolated from CFKO mice showed increased length but not width. The myocardium of CFKO mice exhibited disorganized myofibrils with increased nonmyofibrillar space filled with swelled mitochondria. Last, decreased tyrosine phosphorylation of FAK substrates p130Cas and paxillin were observed in CFKO mice compared with the control littermates. Together, these results provide strong evidence for a role of FAK in the regulation of heart hypertrophy in vivo.

Roles of PPARs on regulating myocardial energy and lipid homeostasis

Myocardial energy and lipid homeostasis is crucial for normal cardiac structure and function. Either shortage of energy or excessive lipid accumulation in the heart leads to cardiac disorders. Peroxisome proliferator-activated receptors (PPARα, -β/δ and -γ), members of the nuclear receptor transcription factor superfamily, play important roles in regulating lipid metabolic genes. All three PPAR subtypes are expressed in cardiomyocytes. PPARα has been shown to control transcriptional expression of key enzymes that are involved in fatty acid (FA) uptake and oxidation, triglyceride synthesis, mitochondrial respiration uncoupling, and glucose metabolism. Similarly, PPARβ/δ is a transcriptional regulator of FA uptake and oxidation, mitochondrial respiration uncoupling, and glucose metabolism. On the other hand, the role of PPARγ on transcriptional regulation of FA metabolism in the heart remains obscure. Therefore, both PPARα and PPARβ/δ are important transcriptional regulators of myocardial energy and lipid homeostasis. Moreover, it appears that the heart needs to have two PPAR subtypes with seemingly overlapping functions in maintaining myocardial lipid and energy homeostasis. Further studies on the potential distinctive roles of each PPAR subtype in the heart should provide new therapeutic targets for treating heart disease.

In Vivo Modeling of Myosin Binding Protein C Familial Hypertrophic Cardiomyopathy

Myosin binding protein C (MyBP-C) is an integral part of the striated muscle sarcomere. As is the case for other sarcomeric genes in human populations, multiple mutations within the gene have been linked to familial hypertrophic cardiomyopathy. Although some MyBP-C lesions are the result of missense mutations, most show truncated polypeptides lacking either the myosin or myosin and titin binding sites. Previously, we generated transgenic (TG) mice with cardiac-specific expression of a MyBP-C mutant lacking the myosin and titin binding domains. Surprisingly, the mutant protein was stable and made up a majority of the MyBP-C species, with concomitant reductions in endogenous MyBP-C such that overall MyBP-C stoichiometry was conserved. In the present study, we created a second series of TG mice that express, in the heart, a mutant MyBP-C lacking only the myosin binding site. In contrast to the previous data for the MyBP-C lacking both titin and myosin binding sites, only very modest levels of protein were found, consistent with data obtained from human biopsies in which mutated MyBP-C could not be detected. Despite normal levels of wild-type MyBP-C, there were significant changes in the structure and ultrastructure of the heart. Fiber mechanics showed decreased unloading shortening velocity, maximum shortening velocity, and relative maximal power output.

Pomegranate flower extract diminishes cardiac fibrosis in zucker diabetic fatty rats : Modulation of cardiac endothelin-1 and nuclear factor-kappaB pathways

The diabetic heart shows increased fibrosis, which impairs cardiac function. Endothelin (ET)-1 and nuclear factor-kappaB (NF-κB) interactively regulate fibroblast growth. We have recently demonstrated that Punica granatum flower (PGF), a Unani anti-diabetic medicine, is a dual activator of peroxisome proliferator-activated receptor (PPAR)-α and -γ, and improves hyperglycemia, hyperlipidemia, and fatty heart in Zucker diabetic fatty (ZDF) rat, a genetic animal model of type 2 diabetes and obesity. Here, we demonstrated that six-week treatment with PGF extract (500 mg/kg, p.o.) in Zucker diabetic fatty rats reduced the ratios of van Gieson-stained interstitial collagen deposit area to total left ventricular area and perivascular collagen deposit areas to coronary artery media area in the heart. This was accompanied by suppression of overexpressed cardiac fibronectin and collagen I and III mRNAs. Punica granatum flower extract reduced the up-regulated cardiac mRNA expression of ET-1, ETA, inhibitor-κBβ and c-jun, and normalized the down-regulated mRNA expression of inhibitor-κBα in Zucker diabetic fatty rats. In vitro, Punica granatum flower extract and its components oleanolic acid, ursolic acid, and gallic acid inhibited lipopolysaccharide-induced NF-κB activation in macrophages. Our findings indicate that Punica granatum flower extract diminishes cardiac fibrosis in Zucker diabetic fatty rats, at least in part, by modulating cardiac ET-1 and NF-κB signaling.

Peroxisome Proliferator-Activated Receptor δ Is an Essential Transcriptional Regulator for Mitochondrial Protection and Biogenesis in Adult Heart

Rationale: Peroxisome proliferator-activated receptors (PPARs) (&agr;, &ggr;, and &dgr;/&bgr;) are nuclear hormone receptors and ligand-activated transcription factors that serve as key determinants of myocardial fatty acid metabolism. Long-term cardiomyocyte-restricted PPAR&dgr; deficiency in mice leads to depressed myocardial fatty acid oxidation, bioenergetics, and premature death with lipotoxic cardiomyopathy. Objective: To explore the essential role of PPAR&dgr; in the adult heart. Methods and Results: We investigated the consequences of inducible short-term PPAR&dgr; knockout in the adult mouse heart. In addition to a substantial transcriptional downregulation of lipid metabolic proteins, short-term PPAR&dgr; knockout in the adult mouse heart attenuated cardiac expression of both Cu/Zn superoxide dismutase and manganese superoxide dismutase, leading to increased oxidative damage to the heart. Moreover, expression of key mitochondrial biogenesis determinants such as PPAR&ggr; coactivator-1 were substantially decreased in the short-term PPAR&dgr; deficient heart, concomitant with a decreased mitochondrial DNA copy number. Rates of palmitate and glucose oxidation were markedly depressed in cardiomyocytes of PPAR&dgr; knockout hearts. Consequently, PPAR&dgr; deficiency in the adult heart led to depressed cardiac performance and cardiac hypertrophy. Conclusions: PPAR&dgr; is an essential regulator of cardiac mitochondrial protection and biogenesis and PPAR&dgr; activation can be a potential therapeutic target for cardiac disorders.

Cardiomyocyte-Specific BMAL1 Plays Critical Roles in Metabolism, Signaling, and Maintenance of Contractile Function of the Heart

Circadian clocks are cell autonomous, transcriptionally based, molecular mechanisms that confer the selective advantage of anticipation, enabling cells/organs to respond to environmental factors in a temporally appropriate manner. Critical to circadian clock function are 2 transcription factors, CLOCK and BMAL1. The purpose of the present study was to reveal novel physiologic functions of BMAL1 in the heart, as well as to determine the pathologic consequences of chronic disruption of this circadian clock component. To address this goal, we generated cardiomyocyte-specific Bmal1 knockout (CBK) mice. Following validation of the CBK model, combined microarray and in silico analyses were performed, identifying 19 putative direct BMAL1 target genes, which included a number of metabolic (e.g., β-hydroxybutyrate dehydrogenase 1 [Bdh1]) and signaling (e.g., the p85α regulatory subunit of phosphatidylinositol 3-kinase [Pik3r1]) genes. Results from subsequent validation studies were consistent with regulation of Bdh1 and Pik3r1 by BMAL1, with predicted impairments in ketone body metabolism and signaling observed in CBK hearts. Furthermore, CBK hearts exhibited depressed glucose utilization, as well as a differential response to a physiologic metabolic stress (i.e., fasting). Consistent with BMAL1 influencing critical functions in the heart, echocardiographic, gravimetric, histologic, and molecular analyses revealed age-onset development of dilated cardiomyopathy in CBK mice, which was associated with a severe reduction in life span. Collectively, our studies reveal that BMAL1 influences metabolism, signaling, and contractile function of the heart.

Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice

Obesity is a calorie‐excessive state associated with high risk of diabetes, atherosclerosis, and certain types of tumors. Obesity may induce inflammation and insulin resistance (IR). We found that the expression of interferon (IFN) regulatory factor 9 (IRF9), a major transcription factor mediating IFN responses, was lower in livers of obese mice than in those of their lean counterparts. Furthermore, whole‐body IRF9 knockout (KO) mice were more obese and had aggravated IR, hepatic steatosis, and inflammation after chronic high‐fat diet feeding. In contrast, adenoviral‐mediated hepatic IRF9 overexpression in both diet‐induced and genetically (ob/ob) obese mice showed markedly improved hepatic insulin sensitivity and attenuated hepatic steatosis and inflammation. We further employed a yeast two‐hybrid screening system to investigate the interactions between IRF9 and its cofactors. Importantly, we identified that IRF9 interacts with peroxisome proliferator‐activated receptor alpha (PPAR‐α), an important metabolism‐associated nuclear receptor, to activate PPAR‐α target genes. In addition, liver‐specific PPAR‐α overexpression rescued insulin sensitivity and ameliorated hepatic steatosis and inflammation in IRF9 KO mice. Conclusion: IRF9 attenuates hepatic IR, steatosis, and inflammation through interaction with PPAR‐α. (Hepatology 2013;58:603–616)

Tumor Suppressor A20 Protects Against Cardiac Hypertrophy and Fibrosis by Blocking Transforming Growth Factor-β–Activated Kinase 1–Dependent Signaling

A20 or tumor necrosis factor–induced protein 3 is a negative regulator of nuclear factor &kgr;B signaling. A20 has been shown previously to attenuate cardiac hypertrophy in vitro and postmyocardial infarction remodeling in vivo. In the present study, we tested the hypothesis that overexpression of A20 in the murine heart would protect against cardiac hypertrophy in vivo. The effects of constitutive human A20 expression on cardiac hypertrophy were investigated using in vitro and in vivo models. Cardiac hypertrophy was produced by aortic banding in A20 transgenic mice and control animals. The extent of cardiac hypertrophy was quantitated by echocardiography, as well as by pathological and molecular analyses of heart samples. Constitutive overexpression of human A20 in the murine heart attenuated the hypertrophic response and markedly reduced inflammation, apoptosis, and fibrosis. Cardiac function was also preserved in hearts with increased A20 levels in response to hypertrophic stimuli. Western blot experiments further showed A20 expression markedly blocked transforming growth factor-&bgr;–activated kinase 1–dependent c-Jun N-terminal kinase/p38 signaling cascade but with no difference in either extracellular signal-regulated kinase 1/2 or AKT activation in vivo and in vitro. In cultured neonatal rat cardiac myocytes, [3H]proline incorporation and Western blot assays revealed that A20 expression suppressed transforming growth factor-&bgr;–induced collagen synthesis and transforming growth factor-&bgr;–activated kinase 1–dependent Smad 2/3/4 activation. In conclusion, A20 improves cardiac functions and inhibits cardiac hypertrophy, inflammation, apoptosis, and fibrosis by blocking transforming growth factor-&bgr;–activated kinase 1–dependent signaling.