"iDoctor – Medical Blog"

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 7 December 2011

Neil Herring, James Cranley, Michael N. Lokale, Dan Li, Julia Shanks, …

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 minutes, in the presence of 10 μM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p < 0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 minutes (n = 6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, all stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n = 6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 minutes (1 μM, n = 5), and completely reversed with the NPY Y2receptor antagonist BIIE 0246 at all time points (1 μM, n = 6). Exogenous galanin (n = 6, 50–500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n = 6). Galanin (500 nM) also significantly attenuated the release ofH-acetylcholine from isolated atria during field stimulation (5 Hz, n = 5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n = 5). Importantly the GalR1receptor was immunofluorescently co-localized with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100 nM, n = 6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibition H89 (500 nM, n = 6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR1receptors on vagal neurons coupled to protein kinase C dependent signaling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.

Highlights

► Galanin is found in guinea pig stellate neurons and GalR1 on cardiac vagal neurons ► Stellate galanin expression increases following 3 days of cell culture ► High level sympathetic stimulation releases galanin which reduces vagal bradycardia ► Galanin reduces acetylcholine release and bradycardia via a GalR1dependent pathway ► Galanin signals via protein kinase C rather than protein kinase A dependent pathways.

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 6 December 2011

Nassiba Merabet, Jeremy Bellien, Etienne Glevarec, Lionel Nicol, Daniele Lucas, …

ObjectivesThe study addressed the hypothesis that soluble epoxide hydrolase (sEH) inhibition, which increases cardiovascular protective epoxyeicosatrienoic acids (EETs), exerts beneficial effects in an established chronic heart failure (CHF) model.MethodsIn CHF rats, left ventricular (LV) function, perfusion and remodeling were assessed using MRI and invasive hemodynamics after 42-days (starting 8 days after coronary ligation) and delayed 3-days (starting 47 days after coronary ligation) treatment with the sEH inhibitor AUDA (twice 0.25 mg/day).ResultsDelayed 3-days and 42-days AUDA increased plasma EETs demonstrating the effective inhibition of sEH. Delayed 3-days and 42-days AUDA enhanced cardiac output without change in arterial pressure, thus reducing total peripheral resistance. Both treatment periods increased the slope of the LV end-systolic pressure-volume relation, but only 42-days AUDA decreased LV end-diastolic pressure, relaxation constant Tau and the slope of the LV end-diastolic pressure-volume relation, associated with a reduced LV diastolic volume and collagen density. Delayed 3-days and, to a larger extent, 42-days AUDA increased LV perfusion associated with a decreased LV hypoxia-inducible factor-1alpha. Both treatment periods decreased reactive oxygen species level and increased reduced-oxidized glutathione ratio. Finally, MSPPOH, an inhibitor of the EETs-synthesizing enzyme cytochrome epoxygenases, abolished the beneficial effects of 3-days AUDA on LV function and perfusion.ConclusionsAugmentation of EETs availability by pharmacological inhibition of sEH increases LV diastolic and systolic function in established CHF. This notably results from short-term processes,i.e.increased LV perfusion, reduced LV oxidative stress and peripheral vasodilatation, but also from long-term effects,i.e.reduced LV remodeling.

Highlights

► We studied the effects of a soluble epoxide hydrolase inhibitor in heart failure rats ► The inhibitor induced an increase in epoxyeicosatrienoic acids bioavailability ► This was associated with an improvement in systolic and diastolic cardiac function ► Enhanced cardiac perfusion and reduced oxidative stress contributed to this effect ► This pharmacological strategy represents a promising approach to treat heart failure

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 2 December 2011

Natasha H. Banke, Lin Yan, Kayla M. Pound, Sunil Dhar, Heather Reinhardt, …

Human studies indicate augmented myocardial lipid metabolism in females, and that sex and obesity interact to predict myocardial fatty acid oxidation and storage. Altered lipid dynamics precede cardiomyopathies, and many studies now address high fat diets. Conversely, caloric restriction (CR), is the most studied model for longevity and stress resistance, including protection against myocardial ischemia. However, no information exists on the effects of long-term caloric restriction (CR) on triacylglyceride (TAG) content and dynamics in the heart. This study explored the effects of CR, sex and age on TAG dynamics in mouse hearts. Male and female SVJ129 mice were fed either normal (ND) or CR diet for 3 or 10 months. In 5-month-old mice, CR similarly decreased cardiac TAG in males (ND: 25.5 +/− 4.5 nanomoles/mg protein; CR: 12.6 +/− 2.7, P < 0.05) and females (ND: 30.1 +/− 4.4; CR: 13.7 +/− 1.2) (no significant differences in TAG content were seen between sexes). CR reduced the contribution of exogenous palmitate to oxidative metabolism in males and females, by 15% and 11% respectively, versus ND, without affecting cardiac workload. CR also induced a larger reduction in TAG turnover in male (68%) than female hearts (38%). Interestingly, in 5 month old male mice, CR reproduced the lower TAG turnover rates of middle-aged males (ND 13-month-old male = 423 +/−76 nanomoles/mg protein/min). Thus, long term CR reduces TAG pool dynamics. Despite reduced content, hearts of female mice subjected to CR retained a more dynamic TAG pool than males, while males respond with greater metabolic remodeling of cardiac lipid dynamics.

Highlights

► The current manuscript compares the effects of gender and age on TAG dynamics in the myocardium after long-term caloric restriction. ► Long-term CR results in a reduction of endogenous lipid stores and a significant decrease in TAG turnover. ► In female myocardium, TAG turnover does not decrease to the same extent as in male myocardium. ► The female myocardium preserves TAG involvement in lipid metabolism.

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 1 December 2011

Feng Hua Yang, W. Glen Pyle

The Z-disc protein CapZ has historically been classified as a structural element, anchoring sarcomeric actin. Our previous work expanded its role to include signal transduction, as CapZ transgenic myofilaments are less sensitive to protein kinase C (PKC). Myocardial PKC has paradoxical effects, mediating both preconditioning and ischemia-reperfusion (IR) injury. Our objective was to determine how decreased CapZ affects IR injury and cardiac preconditioning. Mouse hearts were subjected to 20 min global ischemia and 60 min reperfusion. Some hearts were preconditioned with intermittent IR (IPC). Left ventricular function was assessed and myocardial tissue collected post-IR for molecular analysis and tissue staining. Post-ischemic function was significantly better and infarct size smaller in CapZ transgenic hearts, as compared to wildtype. IPC decreased IR damage in both wildtype and CapZ transgenic hearts, although CapZ transgenic hearts performed significantly better than wildtype. Immunoblotting revealed increased myofilament-associated PKC-α and -ε following IR in wildtype hearts, but no change in PKC-δ or -ζ. By contrast, post-IR myofilament-associated PKC-α was significantly higher in CapZ transgenic mice but the rise in PKC-ε was attenuated. Both PKC-δ and PKC-ζ decreased in CapZ transgenic myofilaments following IR. IPC increased myofilament-associated PKC-α and -ε, while decreasing PKC-δ in wildtype hearts. Preconditioned CapZ IPC hearts showed attenuated increases in myofilament PKC-α and -ε, but also a significant decrease in myofilament PKC-δ and -ζ. These data demonstrate significant differences in post-IR myofilament PKC in untreated and preconditioned CapZ transgenic mice. CapZ reduction did not dramatically affect post-IR myofilament function, nor did preconditioning. These results demonstrate that CapZ deficiency decreases IR injury, while providing enhanced cardioprotection with IPC. The cardioprotected phenotype of CapZ transgenic mice is associated with an altered translocation of PKC-isoforms to cardiac myofilaments.

Highlights

► We examined post-ischemic heart function in CapZ transgenic mice ► CapZ transgenic mice were subjected to acute ischemia-reperfusion ► Reduced CapZ protein increased post-ischemic function compared to wildtype ► Protein kinase C activation was altered in CapZ transgenic mice

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 1 December 2011

Eun Young Kim, Li Chen, Yanlin Ma, Wei Yu, Jiang Chang, …

Sumoylation is a posttranslational modification implicated in a variety of cellular activities, and its role in a number of human pathogeneses such as cleft lip/palate has been well documented. However, the importance of the SUMO conjugation pathway in cardiac development and functional disorders is newly emerging. We previously reported that knockout of SUMO-1 in mice led to congenital heart diseases (CHDs). To further investigate the effects of imbalanced SUMO conjugation on heart development and function and its underlying mechanisms, we generated transgenic (Tg) mice with cardiac-specific expression of SENP2, a SUMO-specific protease that deconjugates sumoylated proteins, to evaluate the impact of desumoylation on heart development and function. Overexpression of SENP2 resulted in premature death of mice with CHDs—atrial septal defects (ASDs) and/or ventricular septal defects (VSDs). Immunobiochemistry revealed diminished cardiomyocyte proliferation in SENP2-Tg mouse hearts compared with that in wild type (WT) hearts. Surviving SENP2-Tg mice showed growth retardation, and developed cardiomyopathy with impaired cardiac function with aging. Cardiac-specific overexpression of the SUMO-1 transgene reduced the incidence of cardiac structural phenotypes in the sumoylation defective mice. Moreover, cardiac overexpression of SENP2 in the mice with Nkx2.5 haploinsufficiency promoted embryonic lethality and severity of CHDs, indicating the functional interaction between SENP2 and Nkx2.5 in vivo. Our findings indicate the indispensability of a balanced SUMO pathway for proper cardiac development and function.

Highlights

► We studied the effect of de-sumoylation on heart development in a transgenic model. ► Increased desumoylation by overexpressed SENP2 in heart caused cardiac defects and dysfunction. ► SENP2 overexpression also caused defect in cardiomyocyte proliferation. ► SUMO-1 overexpression rescued cardiac structural phenotypes in SENP2-Tg mice. ► Balanced SUMO conjugation is essential for normal heart development and function.

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 1 December 2011

Gregory T. Bass, Karen A. Ryall, Ashwin Katikapalli, Brooks E. Taylor, Stephen T. Dang, …

Cardiac hypertrophy is controlled by a complex signal transduction and gene regulatory network, containing multiple layers of crosstalk and feedback. While numerous individual components of this network have been identified, understanding how these elements are coordinated to regulate heart growth remains a challenge. Past approaches to measure cardiac myocyte hypertrophy have been manual and often qualitative, hindering the ability to systematically characterize the network’s higher-order control structure and identify therapeutic targets. Here, we develop and validate an automated image analysis approach for objectively quantifying multiple hypertrophic phenotypes from immunofluorescence images. This approach incorporates cardiac myocyte-specific optimizations and provides quantitative measures of myocyte size, elongation, circularity, sarcomeric organization, and cell-cell contact. As a proof-of-concept, we examined the hypertrophic response to α-adrenergic, β-adrenergic, tumor necrosis factor (TNFα), insulin-like growth factor-1 (IGF-1), and fetal bovine serum pathways. While all five hypertrophic pathways increased myocyte size, other hypertrophic metrics were differentially regulated, forming a distinct phenotype signature for each pathway. Sarcomeric organization was uniquely enhanced by α-adrenergic signaling. TNFα and α-adrenergic pathways markedly decreased cell circularity due to increased myocyte protrusion. Surprisingly, adrenergic and IGF-1 pathways differentially regulated myocyte-myocyte contact, potentially forming a feed-forward loop that regulates hypertrophy. Automated image analysis unlocks a range of new quantitative phenotypic data, aiding dissection of the complex hypertrophic signaling network and enabling myocyte-based high-content drug screening.

Highlights

► Automated image analysis method for cardiac myocyte hypertrophy ► New quantitative measure of sarcomeric organization ► TNFα and α-adrenergic signaling have opposite effects on myocyte elongation. ► Sarcomeric organization is uniquely enhanced by α-adrenergic signaling. ► α- and β-adrenergic pathways enhance myocyte-myocyte contact.

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 1 December 2011

Kobra Haghighi, Tracy Pritchard, Julie Bossuyt, Jason R. Waggoner, Qunying Yuan, …

Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes,similar to PLN-KO,and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a.Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman.Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO)coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 29 November 2011

Takeshi Onizuka, Shinsuke Yuasa, Dai Kusumoto, Kenichiro Shimoji, Toru Egashira, …

The efficient induction of cardiomyocyte differentiation from embryonic stem (ES) cells is crucial for cardiac regenerative medicine. Although Wnts play important roles in cardiac development, complex questions remain as to when, how and what types of Wnts are involved in cardiogenesis. We found that Wnt2 was strongly up-regulated during cardiomyocyte differentiation from ES cells. Therefore, we investigated when and how Wnt2 acts in cardiogenesis during ES cell differentiation. Wnt2 was strongly expressed in the early developing murine heart. We applied this embryonic Wnt2 expression pattern to ES cell differentiation, to elucidate Wnt2 function in cardiomyocyte differentiation. Wnt2 knockdown revealed that intrinsic Wnt2 was essential for efficient cardiomyocyte differentiation from ES cells. Moreover, exogenous Wnt2 increased cardiomyocyte differentiation from ES cells. Interestingly, the effects on cardiogenesis of intrinsic Wnt2 knockdown and exogenous Wnt2 addition were temporally restricted. During cardiomyocyte differentiation from ES cells, Wnt2 didn’t activate canonical Wnt pathway but utilizes JNK/AP-1 pathway which is required for cardiomyocyte differentiation from ES cells. Therefore we conclude that Wnt2 plays strong positive stage-specific role in cardiogenesis through non-canonical Wnt pathway in murine ES cells.

Highlights

► The effects on cardiogenesis of intrinsic Wnt2 were temporally restricted. ► During cardiac differentiation, Wnt2 didn’t activate canonical pathway but JNK/AP-1 pathway. ► Wnt2 plays strong positive stage-specific role in cardiogenesis.

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 24 November 2011

Xi Shi, Chen Yan, Sergiy M. Nadtochiy, Jun-Ichi Abe, Paul S. Brookes, …

Publication year: 2011
Source: Journal of Molecular and Cellular Cardiology, Available online 22 November 2011

Deborah M. Dickey, Daniel L. Dries, Kenneth B. Margulies, Lincoln R. Potter

Cardiomyocytes release atrial natriuretic peptide (ANP) and B-type natriuretic peptide to stimulate processes that compensate for the failing heart by activating guanylyl cyclase (GC)-A. C-type natriuretic peptide is also elevated in the failing heart and inhibits cardiac remodeling by activating the homologous receptor, GC-B. We previously reported that GC-A is the most active membrane GC in normal mouse ventricles while GC-B is the most active membrane GC in failing ventricles due to increased GC-B and decreased GC-A activities. Here, we examined ANP and CNP-specific GC activity in membranes obtained from non-failing and failing human left ventricles and in membranes from matched cardiomyocyte-enriched pellet preparations. Similar to our findings in the murine study, we found that CNP-dependent GC activity was about half of the ANP-dependent GC activity in the non-failing ventricular and was increased in the failing ventricle. ANP and CNP increased GC activity 9- and 5-fold in non-failing ventricles, respectively. In contrast to the mouse study, in failing human ventricles, ANP-dependent activity was unchanged compared to non-failing values whereas CNP-dependent activity increased 35% (p = 0.005). Compared with ventricular membranes, basal GC activity was reduced an order of magnitude in membranes derived from myocyte-enriched pellets from non-failing ventricles. ANP increased GC activity 2.4-fold but CNP only increased GC activity 1.3-fold. In contrast, neither ANP nor CNP increased GC activity in equivalent preparations from failing ventricles. We conclude that: 1) GC-B activity is increased in non-myocytes from failing human ventricles, possibly as a result of increased fibrosis, 2) human ventricular cardiomyocytes express low levels of GC-A and much lower levels or possibly no GC-B, and 3) GC-A in cardiomyocytes from failing human hearts is refractory to ANP stimulation.

Highlights

► Membranes from human heart left ventricles express half as much GC-B activity as GC-A. ► In failing human ventricles, CNP-GC-B-dependent activity was significantly increased. ► Human cardiomyocytes express GC-A but not GC-B. ► GC-A is expressed at reduced levels compared to expression in other cells in the ventricle. ► GC-A in cardiomyocyte-enriched samples from failing human hearts is refractory to ANP.