Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis.

Corticosteroids directly affect the heart and vasculature and are implicated in the pathogenesis of heart failure. Attention is focussed upon the role of the mineralocorticoid receptor (MR) in mediating pro-fibrotic and other adverse effects of corticosteroids upon the heart. In contrast, the role of the glucocorticoid receptor (GR) in the heart and vasculature is less well understood. We addressed this in mice with cardiomyocyte and vascular smooth muscle deletion of GR (SMGRKO mice). Survival of SMGRKO mice to weaning was reduced compared with that of littermate controls. Doppler measurements of blood flow across the mitral valve showed an elongated isovolumetric contraction time in surviving adult SMGRKO mice, indicating impairment of the initial left ventricular contractile phase. Although heart weight was elevated in both genders, only male SMGRKO mice showed evidence of pathological cardiomyocyte hypertrophy, associated with increased myosin heavy chain-ß expression. Left ventricular fibrosis, evident in both genders, was associated with elevated levels of mRNA encoding MR as well as proteins involved in cardiac remodelling and fibrosis. However, MR antagonism with spironolactone from birth only modestly attenuated the increase in pro-fibrotic gene expression in SMGRKO mice, suggesting that elevated MR signalling is not the primary driver of cardiac fibrosis in SMGRKO mice, and cardiac fibrosis can be dissociated from MR activation. Thus, GR contributes to systolic function and restrains normal cardiac growth, the latter through gender-specific mechanisms. Our findings suggest the GR:MR balance is critical in corticosteroid signalling in specific cardiac cell types.

Mineralocorticoid Excess or Glucocorticoid Insufficiency: Renal and Metabolic Phenotypes in a Rat Hsd11b2 Knockout Model.

Obesity and hypertension are 2 major health issues of the 21st century. The syndrome of apparent mineralocorticoid excess is caused by deficiency of 11ß-hydroxysteroid dehydrogenase type 2 (Hsd11b2), which normally inactivates glucocorticoids, rendering the mineralocorticoid receptor aldosterone-specific. The metabolic consequences of Hsd11b2 knockout in the rat are investigated in parallel with electrolyte homeostasis. Hsd11b2 was knocked out, by pronuclear microinjection of targeted zinc-finger nuclease mRNAs, and 1 line was characterized for its response to renal and metabolic challenges. Plasma 11-dehydrocorticosterone was below detection thresholds, and Hsd11b2 protein was undetected by Western blot, indicating complete ablation. Homozygotes were 13% smaller than wild-type littermates, and were polydipsic and polyuric. Their kidneys, adrenals, and hearts were significantly enlarged, but mesenteric fat pads and liver were significantly smaller. On a 0.3% Na diet, mean arterial blood pressure was ≈65 mm Hg higher than controls but only 25 mm Hg higher on a 0.03% Na(+) diet. Urinary Na/K ratio of homozygotes was similar to controls on 0.3% Na(+) diet but urinary albumin and calcium were elevated. Corticosterone and aldosterone levels showed normal circadian variation on both a 0.3% and 0.03% Na(+) diet, but plasma renin was suppressed in homozygotes on both diets. Plasma glucose responses to an oral glucose challenge were reduced despite low circulating insulin, indicating much greater sensitivity to insulin in homozygotes. The rat model reveals mechanisms linking electrolyte homeostasis and metabolic control through the restriction of Hsd11b1 substrate availability.

Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity.

Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) ß, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells.

Functional hypervariability and gene diversity of cardioactive neuropeptides.

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

The effects of membrane potential, SR Ca2+ content and RyR responsiveness on systolic Ca2+ alternans in rat ventricular myocytes.

Previous work has shown that small depolarizing pulses produce a beat to beat alternation in the amplitude of the systolic Ca(2+) transient in ventricular myocytes. The aim of the present work was to investigate the role of changes of SR Ca(2+) content and L-type Ca(2+) current in this alternans. As the amplitude of the depolarizing pulse was increased from 10 to 30 mV the magnitude of alternans decreased. Confocal linescan studies showed that this was accompanied by an increase in the number of sites from which Ca(2+) waves propagated. A sudden decrease in the depolarisation amplitude resulted in three classes of behaviour: (1) a gradual decrease in Ca(2+) transient amplitude before alternans developed accompanied by a loss of SR Ca(2+), (2) a gradual increase in Ca(2+) transient amplitude before alternans accompanied by a gain of SR Ca(2+), and (3) immediate development of alternans with no change of SR content. We conclude that alternans develops if the combination of decreased opening of L-type channels and change of SR Ca(2+) content results in spatially fragmented release from the SR as long as there is sufficient Ca(2+) in the SR to sustain wave propagation. Potentiation of the opening of the ryanodine receptor (RyR) by low concentrations of caffeine (100 microm) abolished alternans for a few pulses but the alternans then redeveloped once SR Ca(2+) content fell to the new threshold for wave propagation. Finally we show evidence that inhibiting L-type Ca(2+) current with 200 mum Cd(2+) produces alternans by means of a similar fragmentation of the Ca(2+) release profile and propagation of mini-waves of Ca(2+) release.

Sarcoplasmic reticulum calcium content fluctuation is the key to cardiac alternans.

The aim of this work was to investigate whether beat-to-beat alternation in the amplitude of the systolic Ca(2+) transient (Ca(2+) alternans) is due to changes of sarcoplasmic reticulum (SR) Ca(2+) content, and if so, whether the alternans arises due to a change in the gain of the feedback controlling SR Ca(2+) content. We found that, in rat ventricular myocytes, stimulating with small (20 mV) depolarizing pulses produced alternans of the amplitude of the Ca(2+) transient. Confocal measurements showed that the larger transients resulted from propagation of Ca(2+) waves. SR Ca(2+) content (measured from caffeine-evoked membrane currents) alternated in phase with the alternans of Ca(2+) transient amplitude. After a large transient, if SR Ca(2+) content was elevated by brief exposure of the cell to a Na(+)-free solution, then the alternans was interrupted and the next transient was also large. This shows that changes of SR Ca(2+) content are sufficient to produce alternans. The dependence of Ca(2+) transient amplitude on SR content was steeper under alternating than under control conditions. During alternation, the Ca(2+) efflux from the cell was also a steeper function of SR Ca(2+) content than under control. We attribute these steeper relationships to the fact that the larger responses in alternans depend on wave propagation and that wave propagation is a steep function of SR Ca(2+) content. In conclusion, alternans of systolic Ca(2+) appears to depend on alternation of SR Ca(2+) content. This, in turn results from the steep dependence on SR Ca(2+) content of Ca(2+) release and therefore Ca(2+) efflux from the cell as a consequence of wave propagation.