Kirsten Hoyer

Mycosporine-like amino acids and phylogenies in green algae : Prasiola and its relatives from the trebouxiophyceae (Chlorophyta)

A UV‐absorbing mycosporine‐like amino acid (324 nm‐MAA), so far only known from the green macroalgal genus Prasiola (Trebouxiophyceae), was also identified in other morphologically diverse green algae closely related to Prasiola spp. in 18S rDNA phylogenies. Using HPLC, a second UV‐absorbing compound was found only in Myrmecia incisa Reisigal among all studied strains. This substance showed an absorption maximum at 322 nm and hence was designated as putative 322 nm‐MAA. Preliminary UV‐exposure experiments indicated that all species containing one or the other MAA showed a strong accumulation of the respective compound, thus supporting their function as putative UV sunscreen. Both UV‐absorbing substances were only identified in the studied members of the Trebouxiophyceae but were absent in members of the Ulvophyceae and Chlorophyceae. When mapped on an 18S rDNA phylogeny, the distribution of 324 nm‐MAA was found to be scattered within the Trebouxiophyceae but was consistent with a distribution that follows phylogenetic patterns rather than ecological adaptations. The 324 nm‐MAA was also detected in two phylogenetically related species from freshwater as well as from subaerial habitats, Watanabea reniformis Hanagata et al. and isolate UR7/5, which were phylogenetically independent of Prasiola and its closer allies. MAAs were absent in another Trebouxiophyceae clade comprising lichen photobionts (Coccomyxa pringsheimii Jaag) as well as freshwater picoplanktonic algae (Choricystis minor (Skuja) Fott). The data presented suggest a chemotaxonomic value of the 324 nm‐MAA in green algal taxonomy. To address the paraphyly of the genus Myrmecia Printz as presently circumscribed, we propose the new combination Lobosphaera incisa.

Rearrangement of energetic and substrate utilization networks compensate for chronic myocardial creatine kinase deficiency

Non‐Technical Summary  Continuous and vigorous heart work is powered by the energetic grid consisting of mitochondria, miniature ATP‐generating fuel cells, and molecular connecting circuits transferring and distributing high‐energy phosphoryls. The creatine kinase (CK) phosphotransfer circuit is the major component of the energetic network, coupling mitochondria with ATP utilization sites, and CK deficiency is a hallmark of cardiovascular diseases. Identification of mechanisms that compensate for reduced CK function would foster approaches leading to recovery and repair of injured hearts. Here, using advanced stable isotope metabolic technologies, we demonstrate that genetic CK deficiency induces a shift in heart energy distribution and substrate utilization networks by redirecting phosphotransfer flux through alternative adenylate kinase, glycolytic and guanine nucleotide systems. Such energetic re‐wiring, together with increased mitochondrial and glycolytic capacities, defines an adaptive metabolomic phenotype of CK deficiency. These findings advance our understanding of cellular energetic infrastructure and provide new perspectives for regulation of energy distribution in disease states.

Cardiac myosin heavy chain isoform exchange alters the phenotype of cTnT-related cardiomyopathies in mouse hearts.

Familial hypertrophic cardiomyopathy, FHC, is a clinically heterogeneous, autosomal-dominant disease of the cardiac sarcomere leading to extensive remodeling at both the whole heart and molecular levels. The remodeling patterns are mutation-specific, a finding that extends to the level of single amino acid substitutions at the same peptide residue. Here we utilize two well-characterized transgenic FHC mouse models carrying independent amino acid substitutions in the TM-binding region of cardiac troponin T (cTnT) at residue 92. R92Q and R92L cTnT domains have mutation-specific average peptide conformation and dynamics sufficient to alter thin filament flexibility and cross-bridge formation and R92 mutant myocytes demonstrate mutation-specific temporal molecular remodeling of Ca(2+) kinetics and impaired cardiac contractility and relaxation. To determine if a greater economy of contraction at the crossbridge level would rescue the mechanical defects caused by the R92 cTnT mutations, we replaced the endogenous murine alpha-myosin heavy chain (MyHC) with the beta-MyHC isoform. While beta-MyHC replacement rescued the systolic dysfunction in R92Q mice, it failed to rescue the defects in diastolic function common to FHC-associated R92 mutations. Surprisingly, a significant component of the whole heart and molecular contractile improvement in the R92Q mice was due to improvements in Ca(2+) homeostasis including SR uptake, [Ca2+](i) amplitude and phospholamban phosphorylation. Our data demonstrate that while genetically altering the myosin composition of the heart bearing a thin filament FHC mutation is sufficient to improve contractility, diastolic performance is refractory despite improved Ca(2+) kinetics. These data reveal a previously unrecognized role for MyHC isoforms with respect to Ca(2+) homeostasis in the setting of cardiomyopathic remodeling and demonstrate the overall dominance of the thin filament mutation in determining the degree of diastolic impairment at the myofilament level.

Reducing the Late Sodium Current Improves Cardiac Function during Sodium Pump Inhibition by Ouabain

Inhibition by cardiac glycosides of Na+, K+-ATPase reduces sodium efflux from myocytes and may lead to Na+ and Ca2+ overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late INa) would reduce ouabains effect to cause cellular Na+ loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na+i) and high-energy phosphates using 23Na and 31P NMR, the amplitude of late INa using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late INa. Ouabain (1–1.3 μM) increased Na+i and late INa of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late INa inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na+i and late INa. Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late INa was also attenuated by the Ca2+-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4′-methoxybenzenesulfonyl)]amino-N-(4′-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na+ and Ca2+ overload is ameliorated by the inhibition of late INa.

Ranolazine Reduces Remodeling of the Right Ventricle and Provoked Arrhythmias in Rats with Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease that often results in right ventricular (RV) failure and death. During disease progression, structural and electrical remodeling of the right ventricle impairs pump function, creates proarrhythmic substrates, and triggers for arrhythmias. Notably, RV failure and lethal arrhythmias are major contributors to cardiac death in patients with PAH that are not directly addressed by currently available therapies. Ranolazine (RAN) is an antianginal, anti-ischemic drug that has cardioprotective effects in experimental and clinical settings of left-sided heart dysfunction. RAN also has antiarrhythmic effects due to inhibition of the late sodium current in cardiomyocytes. We therefore hypothesized that RAN could reduce the maladaptive structural and electrical remodeling of the right ventricle and could prevent triggered ventricular arrhythmias in the monocrotaline rat model of PAH. Indeed, in both in vivo and ex vivo experimental settings, chronic RAN treatment reduced electrical heterogeneity (right ventricular-left ventricular action potential duration dispersion), shortened heart-rate corrected QT intervals in the right ventricle, and normalized RV dysfunction. Chronic RAN treatment also dose-dependently reduced ventricular hypertrophy, reduced circulating levels of B-type natriuretic peptide, and decreased the expression of fibrotic markers. In addition, the acute administration of RAN prevented isoproterenol-induced ventricular tachycardia/ventricular fibrillation and subsequent cardiovascular death in rats with established PAH. These results support the notion that RAN can improve the electrical and functional properties of the right ventricle, highlighting its potential benefits in the setting of RV impairment.

Selective inhibition of the late sodium current has no adverse effect on electrophysiological or contractile function of the normal heart.

Abstract: Inhibition of cardiac late Na+ current (INa,L) decreases sodium-dependent calcium overload in diseased hearts. Because INa,L is small in the absence of disease, its inhibition is not expected to significantly alter function of the normal heart. To test this hypothesis, we determined the effects of GS-458967 (GS967), a novel selective inhibitor of INa,L (IC50 = 0.13 &mgr;M), on cardiac function and hemodynamics. The bradycardic agent ivabradine and the Na+ channel blocker flecainide were used for comparison. A single per os administration of GS967 (5 mg/kg) had no effect on blood pressure or heart rate (HR) in unanesthetized rats. In anesthetized rats, GS967 (0.6 ± 0.1 &mgr;M plasma concentration) had no significant effect on HR, PR or QRS electrocardiogram intervals, or contraction. Flecainide (8 mg/kg) slowed HR by 23% ± 3% (P < 0.001), prolonged the PR and QRS intervals by 42% ± 8% and 64% ± 12% (P < 0.001), and had a significant negative inotropic effect. Ivabradine (3 mg/kg) slowed HR by 36% ± 6% (P < 0.001). In rat and rabbit isolated perfused hearts, GS967 (0.1–3 &mgr;M) had no significant effects on HR, QRS interval, or contractile function. The results show that selective inhibition of cardiac INa,L is not associated with chronotropic, dromotropic, inotropic, or hemodynamic changes.

Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific

•  Familial hypertrophic cardiomyopathy (FHC)‐associated missense mutations in the tail portion of cardiac troponin T (cTnT) lead to a phenotype characterized by an increased cost of cardiac contraction, contractile dysfunction and impaired energetics. •  Arginine 92 (R92) in cTnT is a ‘hotspot’ with many FHC‐associated missense mutations; each mutation leads to a clinically distinct cardiomyopathy. Both R92Q and R92L cTnT mutant mouse hearts exhibit the FHC phenotype. •  Here we found that genetically remodelling the sarcomere by substituting αα‐myosin heavy chain (αα‐MyHC) with ββ‐MyHC normalizes the increased cost of cardiac contraction, rescuing both contractile dysfunction and energetic abnormalities, in R92Q cTnT mutant hearts, while wild‐type R92 and R92L cTnT mutant hearts were unaffected. •  Our results demonstrate that the conformation of the tropomyosin‐binding domain of cTnT induced by each unique amino acid substitution at R92 is a major determinant of sarcomere function.