Background Carnitine palmitoyltransferase 1(CPT1) is a rate-limiting step of mitochondrial β-oxidation by controlling the mitochondrial uptake of long-chain acyl-CoAs. The muscle isoform, CPT1b, is the predominant isoform expressed in the heart. It has been suggested that inhibiting CPT-1 activity by specific CPT-1 inhibitors exerts protective effects against cardiac hypertrophy and heart failure. However, clinical and animal studies have shown mixed results, thereby posting concerns on the safety of this class of drugs. Preclinical studies using genetically modified animal models should provide a better understanding of targeting CPT1 in order to evaluate it as a safe and effective therapeutic approach. Methods and Results Heterozygous CPT1b knockout mice (CPT1b+/−) were subjected to transverse aorta constriction (TAC)-induced pressure-overload. These mice showed overtly normal cardiac structure/function under the basal condition. Under a severe pressure-overload condition induced by two weeks of transverse aorta constriction (TAC), CPT1b+/− mice were susceptible to premature death with congestive heart failure. Under a milder pressure-overload condition, CPT1b+/− mice exhibited exacerbated cardiac hypertrophy and remodeling compared with that in wild-type littermates. There were more pronounced impairments of cardiac contraction with greater eccentric cardiac hypertrophy in CPT1b+/− than in controlled mice. Moreover, the CPT1b+/− heart exhibited exacerbated mitochondrial abnormalities and myocardial lipid accumulation with elevated triglycerides and ceramide content, leading to greater cardiomyocytes apoptosis. Conclusions We conclude that CPT1b deficiency can cause lipotoxicity in the heart under pathological stress, leading to exacerbation of cardiac pathology. Therefore, caution should be applied in the clinical use of CPT-1 inhibitors.
Zebrafish can efficiently regenerate their heart through cardiomyocyte proliferation. In contrast, mammalian cardiomyocytes stop proliferating shortly after birth, limiting the regenerative capacity of the postnatal mammalian heart. Therefore, if the endogenous potential of postnatal cardiomyocyte proliferation could be enhanced, it could offer a promising future therapy for heart failure patients. Here, we set out to systematically identify small molecules triggering postnatal cardiomyocyte proliferation. By screening chemical compound libraries utilizing a Fucci-based system for assessing cell cycle stages, we identified carbacyclin as an inducer of postnatal cardiomyocyte proliferation. In vitro, carbacyclin induced proliferation of neonatal and adult mononuclear rat cardiomyocytes via a peroxisome proliferator-activated receptor δ (PPARδ)/PDK1/p308Akt/GSK3β/β-catenin pathway. Inhibition of PPARδ reduced cardiomyocyte proliferation during zebrafish heart regeneration. Notably, inducible cardiomyocyte-specific overexpression of constitutively active PPARδ as well as treatment with PPARδ agonist after myocardial infarction in mice induced cell cycle progression in cardiomyocytes, reduced scarring, and improved cardiac function. Collectively, we established a cardiomyocyte proliferation screening system and present a new drugable target with promise for the treatment of cardiac pathologies caused by cardiomyocyte loss.
Adiponectin, an adipocyte-derived hormone, is a versatile player involved in the regulation of energy homeostasis, cardiovascular disease, and diabetes. Within adipocytes, adiponectin is retained in the lumen of the endoplasmic reticulum (ER) by binding to the thiol protein ER resident protein 44 kDa (ERp44), which is apparently regulated by the activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma. However, the precise role of ERp44 in adiponectin secretion remains elusive. In the present study, we investigated the functional correlation between ERp44 and adiponectin in a pig model. The transcription of porcine ERp44 was regulated by PPARgamma, which was consistent with the finding of putative peroxisome proliferator response element sites within ERp44 promoter. Using chromatin immunoprecipitation and luciferase reporter assays, we demonstrated that the transcription of porcine ERp44 is repressed through binding of PPARgamma to a peroxisome proliferator response element site located between positions -981 and -1004 in its 5'-flanking region. In human embryonic kidney 293 cells stably transfected with cDNA encoding porcine adiponectin, the secretion of adiponectin was significantly up-regulated and the ERp44 mRNA was down-regulated observably, by either the treatment of PPARgamma agonist rosiglitazone or the overexpression of PPARgamma in these cells. Taken together, our results indicated that PPARgamma is an essential regulatory factor for the transcriptional activity of ERp44, which in turn controls the secretion of adiponectin.
Honokiol is a key component of a medicinal herb, Magnolia bark. Honokiol possesses potential pharmacological benefits for many disease conditions, especially cancer. Recent studies demonstrate that Honokiol exerts beneficial effects on cardiac hypertrophy and doxorubicin (Dox)-cardiotoxicity via deacetylation of mitochondrial proteins. However, the effects and mechanisms of Honokiol on cardiac mitochondrial respiration remain unclear. In the present study, we investigate the effect of Honokiol on cardiac mitochondrial respiration in mice subjected to Dox treatment. Oxygen consumption in freshly isolated mitochondria from mice treated with Honokiol showed enhanced mitochondrial respiration. The Dox-induced impairment of mitochondrial respiration was less pronounced in honokiol-treated than control mice. Furthermore, Luciferase reporter assay reveals that Honokiol modestly increased PPARγ transcriptional activities in cultured embryonic rat cardiomyocytes (H9c2). Honokiol upregulated the expression of PPARγ in the mouse heart. Honokiol repressed cardiac inflammatory responses and oxidative stress in mice subjected to Dox treatment. As a result, Honokiol alleviated Dox-cardiotoxicity with improved cardiac function and reduced cardiomyocyte apoptosis. We conclude that Honokiol protects the heart from Dox-cardiotoxicity via improving mitochondrial function by not only repressing mitochondrial protein acetylation but also enhancing PPARγ activity in the heart. This study further supports Honokiol as a promising therapy for cancer patients receiving Dox treatment.
The Kruppel-like factors (KLFs) belong to the family of zinc finger-containing transcription factors that regulates a diverse array of cellular processes, including cell proliferation, differentiation, and apoptosis. Here we reported the structure, mapping and phylogenetic analysis of KLF gene family in pigs. Comparative analyses revealed strong conservation between pig and human KLFs at the genomic and protein structure levels. Porcine KLF 1-17 were dispersedly located on chromosomes 1, 2, 3, 4, 6, 7, 8, 10, 11, 15, 18 and X, respectively. Based on the phylogenetic analysis, we proposed that KLFs have undergone extensive expansion over the course of evolution. Finally, we identified a characteristic motif in KLF zinc finger domains that can be used to accurately predict potential KLF proteins. The current work represents the first comprehensive study of KLF genes in pigs and provides a foundation for future studies concerning structural, functional, and evolutionary analyses of KLF gene family.
Mitochondrial ATP synthase catalyzes the coupling of oxidative phosphorylation. Under pathological conditions, ATP synthase hydrolyzes ATP to replenish protons from the matrix into the intermembrane space, sustaining mitochondrial membrane potential. ATPase inhibitory factor 1 (IF1) is a nuclear-encoded, ATP synthase-interacting protein that selectively inhibits the hydrolysis activity of ATP synthase, which may render the protective role of IF1 in ischemic hearts. However, the in vivo cardiac function of IF1 and the potential therapeutic application targeting IF1 remain obscure. In the present study, we uncovered that IF1 is upregulated in mouse hearts with pressure overload-induced hypertrophy and in human hearts with dilated cardiomyopathy. IF1 knockout (KO) mice were protected against cardiac dysfunction and pathological development induced by transverse aortic constriction (TAC) or isoproterenol infusion. The reduced ATP hydrolysis activated AMPK activity in IF1 KO hearts, which together facilitated autophagy. These results suggest that IF1 upregulation in the failing heart may be a maladaptive response. Inhibiting IF1 in the hypertrophied heart not only prevents cell death from excessive mitochondrial depolarization but also activates AMPK signaling and increases autophagy. Therefore, IF1 inhibition may serve as a potential therapeutic target in treating pathological cardiac hypertrophy and heart failure.
The hormone resistin, which was originally shown to induce insulin resistance, has been implicated in the regulation of inflammatory processes, but the molecular mechanism underlying such regulation has not been clearly defined. The goal of our study was to determine whether the expression of COX-2 can be induced by resistin and what the potential signaling pathway involved in this process is. Compared with controls, resistin significantly upregulated COX-2 expression in RAW264.7 macrophage cells. Administration of anti-resistin antibody could significantly reduce this effect. Induction of COX-2 by resistin was also markedly reduced in the presence of either dominant negative mutant IkappaBalpha or PDTC, a pharmacological inhibitor of NF-kappaB. On the other hand, NF-kappaB subunit p65 was upregulated by resistin. Moreover, we found that transforming growth factor-beta-activated kinase 1 (TAK1), a mitogen-activated protein kinase kinase kinase (MAPKKK), could be activated in response to resistin. These results suggest that resistin enhances COX-2 expression in mouse macrophage cells in a TAK1-IKK-NF-kappaB-dependent manner and therefore plays a critical role in inflammatory processes.
In this chapter, we describe detailed protocols for measuring high-resolution respirometry on mitochondria extracted from adult whole mouse heart using the Oroboros 2k-Oxygraph system. The method provides detailed procedures for the preparation of mitochondria and measurement of high-resolution respirometry in response to various respiration inhibitions. The method described in this chapter could discern the different respiration rate on mitochondria extracted from two spatially distinct mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM). These approaches can easily be translated to other cells and tissues.
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