Maintenance Of Contractile Function Research Articles

P300 and cAMP response element-binding protein-binding protein in skeletal muscle homeostasis, contractile function, and survival.

BackgroundReversible ε‐amino acetylation of lysine residues regulates transcription as well as metabolic flux; however, roles for specific lysine acetyltransferases in skeletal muscle physiology and function are unknown. In this study, we investigated the role of the related acetyltransferases p300 and cAMP response element‐binding protein‐binding protein (CBP) in skeletal muscle transcriptional homeostasis and physiology in adult mice.MethodsMice with skeletal muscle‐specific and inducible knockout of p300 and CBP (PCKO) were generated by crossing mice with a tamoxifen‐inducible Cre recombinase expressed under the human α‐skeletal actin promoter with mice having LoxP sites flanking exon 9 of the Ep300 and Crebbp genes. Knockout of PCKO was induced at 13–15 weeks of age via oral gavage of tamoxifen for 5 days to both PCKO and littermate control [wildtype (WT)] mice. Body composition, food intake, and muscle function were assessed on day 0 (D0) through 5 (D5). Microarray and tandem mass tag mass spectrometry analyses were performed to assess global RNA and protein levels in skeletal muscle of PCKO and WT mice.ResultsAt D5 after initiating tamoxifen treatment, there was a reduction in body weight (−15%), food intake (−78%), stride length (−46%), and grip strength (−45%) in PCKO compared with WT mice. Additionally, ex vivo contractile function [tetanic tension (kPa)] was severely impaired in PCKO vs. WT mice at D3 (~70–80% lower) and D5 (~80–95% lower) and resulted in lethality within 1 week—a phenotype that is reversed by the presence of a single allele of either p300 or CBP. The impaired muscle function in PCKO mice was paralleled by substantial transcriptional alterations (3310 genes; false discovery rate < 0.1), especially in gene networks central to muscle contraction and structural integrity. This transcriptional uncoupling was accompanied by changes in protein expression patterns indicative of impaired muscle function, albeit to a smaller magnitude (446 proteins; fold‐change > 1.25; false discovery rate < 0.1).ConclusionsThese data reveal that p300 and CBP are required for the control and maintenance of contractile function and transcriptional homeostasis in skeletal muscle and, ultimately, organism survival. By extension, modulating p300/CBP function may hold promise for the treatment of disorders characterized by impaired contractile function in humans.

Kruppel-like factor 15 is required for the cardiac adaptive response to fasting.

Cardiac metabolism is highly adaptive in response to changes in substrate availability, as occur during fasting. This metabolic flexibility is essential to the maintenance of contractile function and is under the control of a group of select transcriptional regulators, notably the nuclear receptor family of factors member PPARα. However, the diversity of physiologic and pathologic states through which the heart must sustain function suggests the possible existence of additional transcriptional regulators that play a role in matching cardiac metabolism to energetic demand. Here we show that cardiac KLF15 is required for the normal cardiac response to fasting. Specifically, we find that cardiac function is impaired upon fasting in systemic and cardiac specific Klf15-null mice. Further, cardiac specific Klf15-null mice display a fasting-dependent accumulation of long chain acylcarnitine species along with a decrease in expression of the carnitine translocase Slc25a20. Treatment with a diet high in short chain fatty acids relieves the KLF15-dependent long chain acylcarnitine accumulation and impaired cardiac function in response to fasting. Our observations establish KLF15 as a critical mediator of the cardiac adaptive response to fasting through its regulation of myocardial lipid utilization.

ENDOTHELIAL DYSFUNCTION AND DEFECTIVE SMOOTH MUSCLE CONTRACTILITY IN ANEURYSMAL FIBULIN-4 DEFICIENT MICE: 8A.05

Objective: Using Fibulin-4 knockdown mice (Fibulin-4R/R), we previously showed that the dosage of Fibulin-4 can determine the severity of aneurysm formation. Strikingly, even a modest reduction in expression of Fibulin-4 in the heterozygous Fibulin-4+/R mice occasionally resulted in small aneurysm formation. Aortic pulse pressure was 2 to 3-fold higher in Fibulin-4R/R mice, resulting from increased aortic stiffness. Here, we analyzed the altered biological pathways in aneurismal disease at the molecular, cellular and functional level. Methods: Aorta transcriptome changes of Fibulin-4+/R and Fibulin-4R/R aortas were performed using ANOVA and Ingenuity Pathway Analysis. Histology was applied to verify cellular abnormalities. Functional analysis occurred by measuring isometric forces of thoracic aortas in vitro and aortic pressures with a pressure transducer catheter in vivo. Results: Ingenuity Pathway Analysis identified three major dysregulated pathways, including TGFβ signaling, immune response and specifically Ca2+ signaling genes involved in the maintenance of contractile function. Histological analysis and á-smooth muscle actin immunostaining showed loss of smooth muscle cells (SMCs), which coincided with cartilage bone formation, already in Fibulin-4+/R animals. Severe loss of SMCs and increase of extracellular matrix depositions resulted in overall thickening of the aortic wall in Fibulin-4R/R mice. In addition, Fibulin-4R/R mice showed increased endothelial damage, endothelial cellular proliferation, as evidenced by BrdU labeling and thrombus formation, indicative for endothelial dysfunction. In vitro, maximum contractility was determined for phenylephrine and compared to KCl (100 mM). KCl responses showed a reduced contractile capacity of ascending aortas in Fibulin-4R/R mice correlating with a reduction of SMCs in this area. In descending aortas, phenylephrine-induced contractility decreased from 54 ± 7% in wildtypes to 13 ± 2% in Fibulin-4R/R mice. Endothelium-dependent vasorelaxation by acetylcholine, following preconstruction with U46619, was strongly reduced in Fibulin-4R/R mice. Finally, with reducing expression of fibulin-4, diastolic blood pressure decreased from 63 ± 3 in Fibulin-4+/+ to 60 ± 5 in Fibulin-4+/R and 41 ± 7 in Fibulin-4R/R mice. Conclusion: Our results uncover a role for Fibulin-4 in the maintenance of SMC contractile function and endothelial responsiveness.

Prox1 maintains muscle structure and growth in the developing heart

Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the murine homeobox transcription factor Prox1 results in the disruption of expression and localisation of sarcomeric proteins, gross myofibril disarray and growth-retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of the genes encoding the structural proteins alpha-actinin, N-RAP and zyxin, which collectively function to maintain an actin-alpha-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and the manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study reveals an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling foetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.

Gene deletional strategies reveal novel physiological roles for myoglobin in striated muscle

Myoglobin is an abundant hemoprotein that is expressed in cardiomyocytes and oxidative skeletal myofibers of vertebrates. Elegant studies using physiological, biochemical and spectroscopic analyses support a role for myoglobin in facilitated oxygen transport and as a reservoir for oxygen in muscle of diving and hypoxia-adapted animals. In contrast, the functional role of myoglobin in terrestrial animals that function at ambient oxygen levels is a subject of debate. This debate was further fueled by the observation that genetically engineered mice that lack myoglobin are viable and capable of withstanding the hemodynamic stress associated with reproduction. Analysis of the myoglobin mutant striated muscle reveals a spectrum of adaptive mechanisms that partially compensate for the absence of myoglobin and further supports an important function for this hemoprotein in the maintenance of contractile function during exercise under ambient and hypoxic conditions. Future studies utilizing transgenic and gene deletional strategies will further enhance our understanding of myoglobin function under normoxic and hypoxic conditions and will impact our understanding of exercise physiology.

Integration of vascular, contractile and metabolic responses to hypoxia: effects of maturation and adenosine.

We examined myocardial responses to reduced arterial PO2 and the role of endogenous adenosine in constant-pressure perfused hearts from immature (Imm) and mature (Mat) rabbits. During normoxia, coronary flow and myocardial O2 consumption were similar in both groups. With moderate hypoxia, coronary perfusate flow increased by 125 +/- 16% in Imm but by only 68 +/- 12% in Mat hearts. Imm hearts displayed better maintenance of contractile function (87 vs. 67% in Mat hearts) and metabolic state. Blockade of cardiac adenosine receptors with 25 microM 8-p-sulfophenyltheophylline attenuated vasodilation during hypoxia, reduced contractile function, and abolished age-related differences in one response to hypoxia. Myocardial purine release and extracellular purine levels were threefold higher in Mat compared with Imm hearts and was associated with higher cytosolic 5'-AMP concentration (and lower [ATP]/[ADP].[P(i)], where [ATP], [ADP], and [P(i)] are concentrations of ATP, ADP and P(i), respectively) in Mat hearts. In summary, 1) Imm hearts are functionally and metabolically more tolerant of hypoxic perfusion, largely because of improved hypoxia-induced coronary vasodilation; 2) endogenous adenosine mediates this beneficial vasodilation, enhancing hypoxic tolerance; and 3) improved vascular sensitivity to adenosine allows for enhanced vasodilatory responses in the face of lower adenosine levels and higher energy state in immature hearts.

Effect of retinoic acid on contractile competence of vascular smooth muscle.

Rat aortic rings cultured for 24 h in protein-free medium showed a significant reduction in the contractile response to potassium (-60%) and to phorbol 12,13-dibutyrate (-65%). The addition of plasma to the medium attenuated the loss in responsiveness, and the supplementation of the plasma-containing medium with all trans-retinoic acid (RA) returned the response to potassium (85%) and phorbol ester (135%) to near normal or supramaximal compared with fresh tissue. Furthermore, the combined additions of plasma and RA resulted in significant preservation of the contractile response (75%) for at least 3 days in organ culture. Removal of the endothelium before organ culture eliminated the enhancement of contraction by plasma and RA. However, the removal of the endothelium from tissues that had been cultured with an intact endothelium or the blockade of nitric oxide synthesis in these tissues before contractile measurements had no effect on the contractile response. Finally, the incubation of tissues with phorbol ester to downregulate protein kinase C resulted in a marked attenuation (-75%) of the contractile response compared with control tissues in culture. The results suggest that circulating factors may be necessary for the maintenance of contractile function of aortic smooth muscle. Based on the opposing actions of RA and phorbol ester, the long-term regulation of contractile function may involve protein kinase C activity.

Changes in citric acid cycle flux and anaplerosis antedate the functional decline in isolated rat hearts utilizing acetoacetate.

To determine the temporal relationship between changes in contractile performance and flux through the citric acid cycle in hearts oxidizing acetoacetate, we perfused isolated working rat hearts with either glucose or acetoacetate (both 5 mM) and freeze-clamped the tissue at defined times. After 60 min of perfusion, hearts utilizing acetoacetate exhibited lower systolic and diastolic pressures and lower cardiac outputs. The oxidation of acetoacetate increased the tissue content of 2-oxoglutarate and glutamate and decreased the content of succinyl-CoA suggesting inhibition of citric acid cycle flux through 2-oxoglutarate dehydrogenase. Whereas hearts perfused with either acetoacetate or glucose were similar with respect to their function for the first 20 min, changes in tissue metabolites were already observed within 5 min of perfusion at near-physiological workloads. The addition of lactate or propionate, but not acetate, to hearts oxidizing acetoacetate improved contractile performance, although inhibition of 2-oxoglutarate dehydrogenase was probably not diminished. If lactate or propionate were added, malate and citrate accumulated indicating utilization of anaplerotic pathways for the citric acid cycle. We conclude that a decreased rate of flux through 2-oxoglutarate dehydrogenase in hearts oxidizing acetoacetate precedes, and may be responsible for, contractile failure and is not the result of decreased cardiac work. Further, anaplerosis play an important role in the maintenance of contractile function in hearts utilizing acetoacetate.

Quantitative analysis of muscle breakdown during starvation in the marine flatfish Pleuronectes platessa.

The present study describes the effects of starvation for a duration of four months on the ultrastructure of skeletal muscles from the marine flatfish (Pleuronectes platessa L.). Starvation is associated with a decrease in resting metabolic rate from 20.1 +/- 2.2 to 11.6 +/- 1.5 mg . O2/kg/h (P less than 0.05) and muscle wasting. Median fibre size fell from 700 micrometer 2 to 500 micrometer 2 in intermediate (fast oxidative) and from 1,800 micrometer 2 to 600 micrometer 2 in starved, white (fast-glycolytic) muscle fibres. In contrast, median fibre size in red (slow oxidative) muscle remained within the range 300-400 micrometer 2. The fraction of red fibre volume occupied by myofibrils (58.6%) and mitochondria (24.5%) did not change significantly with starvation. There was, however, a decrease in stored lipid 110.7% to 3.2%) and an alteration in the structure of the cristae in mitochondria from red muscle. Atrophy of white muscle fibres is associated with a decrease in both the diameter and fractional volume occupied by myofibrils (85.7% to 61.9% P less than 0.01). In a high proportion of white fibres peripheral degeneration of Z-discs is evident causing an unravelling of the thin filament lattice. It is suggested that this allows a partial decrease in myofibril diameter and hence the maintenance of contractile function in muscle from starved fish. In severely degenerating white fibres, disorganised thick and thin filaments and numerous multi-membrane lysosome-like vesicles are observed. Starvation results in an increase in the average content of mitochondria in white fibres from 2.2 to 6.7% (P less than 0.01). In fed plaice mitochondria constitute less than 1% of the volume of the white fibre in 43.5% of the fibres. The proportion of white fibres containing more than 6% mitochondria increases from 6.5% to 58% with starvation.

Removal of sialic acid from cardiac sarcolemma does not affect contractile function in electrically stimulated guinea pig left atria.

Calcium is essential for the maintenance of contraction in the heart, and it has been suggested that the glycoprotein matrix on the external surface of cardiac cells is a critical factor in the supply of 'activator' calcium to the beating heart. Sialic acid residues are important calcium-binding sites in the matrix and treatment with neuraminidase, an enzyme which cleaves sialic acid from oligosaccharide chains, has been reported to abolish spontaneous contraction in cultured heart cells, without causing general breakdown in the plasma membrane. However, we report here that in intact, electrically stimulated guinea pig atrial preparations, neuraminidase treatment produces no significant changes in resting tension or force of contraction. It is also without effect on the response to calcium removal and replacement, and inotropic or toxic concentrations of cardiac glycosides. In these experiments, up to 79% of the total tissue sialic acid was removed, and electron microscopy studies showed that this removal was accompanied by marked reduction of lanthanum binding to the sarcolemma. We therefore conclude that the calcium-binding sialic acid residues in the external matrix of the cardiac cells are of little importance in the maintenance of contractile function in these intact atrial preparations.