DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations.

RATIONALE: Mutations in the LMNA gene, encoding LMNA (lamin A/C), are responsible for laminopathies. Dilated cardiomyopathy (DCM) is a major cause of mortality and morbidity in laminopathies. OBJECTIVE: To gain insights into the molecular pathogenesis of DCM in laminopathies. METHODS AND RESULTS: We generated a tet-off bigenic mice expressing either a WT (wild type) or a mutant LMNA (D300N) protein in cardiac myocytes. LMNAD300N mutation is associated with DCM in progeroid syndromes. Expression of LMNAD300N led to severe myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. Administration of doxycycline suppressed LMNAD300N expression and prevented the phenotype. Whole-heart RNA sequencing in 2-week-old WT and LMNAD300N mice led to identification of ≈6000 differentially expressed genes. Gene Set Enrichment and Hallmark Pathway analyses predicted activation of E2F (E2F transcription factor), DNA damage response, TP53 (tumor protein 53), NFκB (nuclear factor κB), and TGFß (transforming growth factor-ß) pathways, which were validated by Western blotting, quantitative polymerase chain reaction of selected targets, and immunofluorescence staining. Differentially expressed genes involved cell death, cell cycle regulation, inflammation, and epithelial-mesenchymal differentiation. RNA sequencing of human hearts with DCM associated with defined LMNA pathogenic variants corroborated activation of the DNA damage response/TP53 pathway in the heart. Increased expression of CDKN2A (cyclin-dependent kinase inhibitor 2A)-a downstream target of E2F pathway and an activator of TP53-provided a plausible mechanism for activation of the TP53 pathway. To determine pathogenic role of TP53 pathway in DCM, Tp53 gene was conditionally deleted in cardiac myocytes in mice expressing the LMNAD300N protein. Deletion of Tp53 partially rescued myocardial fibrosis, apoptosis, proliferation of nonmyocyte cells, left ventricular dilatation and dysfunction, and slightly improved survival. CONCLUSIONS: Cardiac myocyte-specific expression of LMNAD300N, associated with DCM, led to pathogenic activation of the E2F/DNA damage response/TP53 pathway in the heart and induction of myocardial fibrosis, apoptosis, cardiac dysfunction, and premature death. The findings denote the E2F/DNA damage response/TP53 axis as a responsible mechanism for DCM in laminopathies and as a potential intervention target.

Influence of dark phase restricted high fat feeding on myocardial adaptation in mice.

Prolonged high fat feeding is associated with myocardial contractile dysfunction in rodents. However, epidemiological data do not necessarily support the concept that fat-enriched diets adversely affect cardiac function in humans. When fed in an ad libitum manner, laboratory rodents consume chow throughout the day. In contrast, humans typically consume food only during the awake phase. Discrepancies between rodent and human feeding behaviors led us to hypothesize that the time of day at which dietary lipids are consumed significantly influences myocardial adaptation. In order to better mimic feeding behavior in humans, mice were fed (either a control or high fat diet) only during the 12-hour dark phase (i.e., no food was provided during the light phase). We report that compared to dark phase restricted control diet fed mice, mice fed a high fat diet during the dark phase exhibit: 1) essentially normal body weight gain and energy balance; 2) increased fatty acid oxidation at whole body, as well as skeletal and cardiac muscle (in the presence of insulin and/or at high workloads) levels; 3) induction of fatty acid responsive genes, including genes promoting triglyceride turnover in the heart; 4) no evidence of cardiac hypertrophy; and 5) persistence/improvement of myocardial contractile function, as assessed ex vivo. These data are consistent with the hypothesis that ingestion of dietary fat only during the more active/awake period allows adequate metabolic adaptation, thereby preserving myocardial contractile function. This article is part of a Special Issue entitled "Focus on cardiac metabolism".

O-GlcNAcylation, novel post-translational modification linking myocardial metabolism and cardiomyocyte circadian clock.

The cardiomyocyte circadian clock directly regulates multiple myocardial functions in a time-of-day-dependent manner, including gene expression, metabolism, contractility, and ischemic tolerance. These same biological processes are also directly influenced by modification of proteins by monosaccharides of O-linked ß-N-acetylglucosamine (O-GlcNAc). Because the circadian clock and protein O-GlcNAcylation have common regulatory roles in the heart, we hypothesized that a relationship exists between the two. We report that total cardiac protein O-GlcNAc levels exhibit a diurnal variation in mouse hearts, peaking during the active/awake phase. Genetic ablation of the circadian clock specifically in cardiomyocytes in vivo abolishes diurnal variations in cardiac O-GlcNAc levels. These time-of-day-dependent variations appear to be mediated by clock-dependent regulation of O-GlcNAc transferase and O-GlcNAcase protein levels, glucose metabolism/uptake, and glutamine synthesis in an NAD-independent manner. We also identify the clock component Bmal1 as an O-GlcNAc-modified protein. Increasing protein O-GlcNAcylation (through pharmacological inhibition of O-GlcNAcase) results in diminished Per2 protein levels, time-of-day-dependent induction of bmal1 gene expression, and phase advances in the suprachiasmatic nucleus clock. Collectively, these data suggest that the cardiomyocyte circadian clock increases protein O-GlcNAcylation in the heart during the active/awake phase through coordinated regulation of the hexosamine biosynthetic pathway and that protein O-GlcNAcylation in turn influences the timing of the circadian clock.

Cardiac-specific mutation of Clock alters the quantitative measurements of physical activities without changing behavioral circadian rhythms.

Even though peripheral circadian oscillators in the cardiovascular system are known to exist, the daily rhythms of the cardiovascular system are mainly attributed to autonomic or hormonal inputs under the control of the central oscillator, the suprachiasmatic nucleus (SCN). In order to examine the role of peripheral oscillators in the cardiovascular system, we used a transgenic mouse where the Clock gene is specifically disrupted in cardiomyocytes. In this cardiomyocyte-specific CLOCK mutant (CCM) mouse model, the circadian input from the SCN remains intact. Both CCM and wild-type (WT) littermates displayed circadian rhythms in wheel-running behavior. However, the overall wheel-running activities were significantly lower in CCM mice compared to WT over the course of 5 weeks, indicating that CCM mice either have lower baseline physical activities or they have lower physical adaptation abilities because daily wheel running, like routine exercise, induces physical adaptation over a period of time. Upon further biochemical analysis, it was revealed that the diurnal oscillations of phosphorylation states of several kinases and protein expression of the L-type voltage-gated calcium channel (L-VGCC) α1D subunit found in WT hearts were abolished in CCM hearts, indicating that in mammalian hearts, the daily oscillations of the activities of these kinases and L-VGCCs were downstream elements of the cardiac core oscillators. However, the phosphorylation of p38 MAPK exhibited robust diurnal rhythms in both WT and CCM hearts, indicating that cardiac p38 could be under the influence of the central clock through neurohormonal signals or be part of the circadian input pathway in cardiomyocytes. Taken together, these results indicate that the cardiac core oscillators have an impact in regulating circadian rhythmicities and cardiac function.

Evidence suggesting that the cardiomyocyte circadian clock modulates responsiveness of the heart to hypertrophic stimuli in mice.

Circadian dyssynchrony of an organism (at the whole-body level) with its environment, either through light-dark (LD) cycle or genetic manipulation of clock genes, augments various cardiometabolic diseases. The cardiomyocyte circadian clock has recently been shown to influence multiple myocardial processes, ranging from transcriptional regulation and energy metabolism to contractile function. The authors, therefore, reasoned that chronic dyssychrony of the cardiomyocyte circadian clock with its environment would precipitate myocardial maladaptation to a circadian challenge (simulated shiftwork; SSW). To test this hypothesis, 2- and 20-month-old wild-type and CCM (Cardiomyocyte Clock Mutant; a model with genetic temporal suspension of the cardiomyocyte circadian clock at the active-to-sleep phase transition) mice were subjected to chronic (16-wks) biweekly 12-h phase shifts in the LD cycle (i.e., SSW). Assessment of adaptation/maladaptation at whole-body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels revealed essentially identical responses between wild-type and CCM littermates. However, CCM hearts exhibited increased biventricular weight, cardiomyocyte size, and molecular markers of hypertrophy (anf, mcip1), independent of aging and/or SSW. Similarly, a second genetic model of selective temporal suspension of the cardiomyocyte circadian clock (Cardiomyocyte-specific BMAL1 Knockout [CBK] mice) exhibits increased biventricular weight and mcip1 expression. Wild-type mice exhibit 5-fold greater cardiac hypertrophic growth (and 6-fold greater anf mRNA induction) when challenged with the hypertrophic agonist isoproterenol at the active-to-sleep phase transition, relative to isoproterenol administration at the sleep-to-active phase transition. This diurnal variation was absent in CCM mice. Collectively, these data suggest that the cardiomyocyte circadian clock likely influences responsiveness of the heart to hypertrophic stimuli.

Direct regulation of myocardial triglyceride metabolism by the cardiomyocyte circadian clock.

Maintenance of circadian alignment between an organism and its environment is essential to ensure metabolic homeostasis. Synchrony is achieved by cell autonomous circadian clocks. Despite a growing appreciation of the integral relation between clocks and metabolism, little is known regarding the direct influence of a peripheral clock on cellular responses to fatty acids. To address this important issue, we utilized a genetic model of disrupted clock function specifically in cardiomyocytes in vivo (termed cardiomyocyte clock mutant (CCM)). CCM mice exhibited altered myocardial response to chronic high fat feeding at the levels of the transcriptome and lipidome as well as metabolic fluxes, providing evidence that the cardiomyocyte clock regulates myocardial triglyceride metabolism. Time-of-day-dependent oscillations in myocardial triglyceride levels, net triglyceride synthesis, and lipolysis were markedly attenuated in CCM hearts. Analysis of key proteins influencing triglyceride turnover suggest that the cardiomyocyte clock inactivates hormone-sensitive lipase during the active/awake phase both at transcriptional and post-translational (via AMP-activated protein kinase) levels. Consistent with increased net triglyceride synthesis during the end of the active/awake phase, high fat feeding at this time resulted in marked cardiac steatosis. These data provide evidence for direct regulation of triglyceride turnover by a peripheral clock and reveal a potential mechanistic explanation for accelerated metabolic pathologies after prevalent circadian misalignment in Western society.

Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression.

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 microM epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.

Receptor internalization in yeast requires the Tor2-Rho1 signaling pathway.

Efficient internalization of proteins from the cell surface is essential for regulating cell growth and differentiation. In a screen for yeast mutants defective in ligand-stimulated internalization of the alpha-factor receptor, we identified a mutant allele of TOR2, tor2G2128R. Tor proteins are known to function in translation initiation and nutrient sensing and are required for cell cycle progression through G1. Yeast Tor2 has an additional role in regulating the integrity of the cell wall by activating the Rho1 guanine nucleotide exchange factor Rom2. The endocytic defect in tor2G2128R cells is due to disruption of this Tor2 unique function. Other proteins important for cell integrity, Rom2 and the cell integrity sensor Wsc1, are also required for efficient endocytosis. A rho1 mutant specifically defective in activation of the glucan synthase Fks1/2 does not internalize alpha-factor efficiently, and fks1Delta cells exhibit a similar phenotype. Removal of the cell wall does not inhibit internalization, suggesting that the function of Rho1 and Fks1 in endocytosis is not through cell wall synthesis or structural integrity. These findings reveal a novel function for the Tor2-Rho1 pathway in controlling endocytosis in yeast, a function that is mediated in part through the plasma membrane protein Fks1.