Changes in cardiac phenotype in hypertrophy and failure: From receptor to gene

The terminally differentiated adult cardiac myocyte cannot undergo cellular division. Growth of the heart in response to chronic hemodynamic overload therefore occurs through hypertrophy of the myocytes. The adaptation of the myocyte during hypertrophy not only involves an increase in cell size but also results in a change in phenotype through modification of the pattern of gene expression. From in vitro studies, it can be learned that agonists like angiotensin-II, endothelin-1, cardiotrophin, basic fibroblast growth factor, insulin-like growth factor-I, or stimulation with the α1-adrenergic agonist phenylephrine can induce hypertrophy. In vivo studies suggest that especially angiotensin-II and endothelin-1 play a prominent role in induction of hypertrophy during overload. These agonists couple to classical seven- transmembrane spanning domain (serpentine) receptors, signaling through activation of the phosphoinositide pathway. This leads to generation of 1,2- diacylglycerol and activation of protein kinase C. Surprisingly, however, these agonists were also shown to activate the mitogen-activated kinase (MAPK) pathway that is typically activated by (growth factor) receptors harboring (intrinsic) tyrosine kinase activity. Increased mechanical forces exerted on the heart during overload also induce hypertrophy, partly through autocrine and paracrine factors such as angiotensin-II and/or endothelin-1. However, direct stimulation of MAPK pathways by stretch might also be exerted through cross-talk with an activated integrin-focal adhesion kinase pathway. Activated MAPK partly translocates to the nucleus, where phosphorylation processes are initiated that lead to altered transcription factor activity. Some transcription factors involved in expression regulation in the hypertrophic myocyte have now been implied, and knowledge concerning genetic cis-acting elements that are involved is also increasing. However, the complexity and interplay of different (and possibly still unknown) signaling pathways do not yet warrant a complete picture regarding the mechanism of in vivo hypertrophy development during overload

Phosphorylation by protein kinase C and the responsiveness of Mg2+-ATPase to Ca2+ of myofibrils isolated from stunned and non-stunned porcine myocardinm

Previously we showed in an in situ porcine model that the thiadiazinone derivative [+]EMD 60263, a Ca2+ sensitizer without phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in non-stunned myocardium. This finding was consistent with the observed leftward shifts of the pCa2+/Mg2+-ATPase curves of isolated myofibrils induced by [+]EMD 60263. The aim of the present investigation was to study the possible involvement of protein kinase C in the mechanism of reduced Ca2+ responsiveness of myofilaments during stunning. No differences were observed in the maximal activity of the Ca2+-stimulated Mg2+-ATPase and in the pCa50 of myofibrils isolated from non-stunned and stunned myocardium. After phosphorylation with [gamma-32P]-ATP and excess of purified rat brain protein kinase C, the myofibrils were separated on sodiumdodecylsulphate-polyacrylamide gelectrophoresis and the 32P incorporation counted by the Molecular Imager. Ca2+/ phosphatidylserine/sn-1,2 diolein-dependent 32P incorporation catalyzed by excess of purified rat brain protein kinase C in C-protein, TnT and TnI subunits did not show any differences between myofibrils from non-stunned and stunned myocardium. However, protein kinase C-induced phosphorylation of myofibrils isolated from ventricular myocardium of sham-operated pigs resulted in a marked leftward shift of the PCa50 from 6.03 ± 0.04 to 6.44 ± 0.06 (p < 0.05), while porcine heart cyclic AMP-dependent protein kinase-induced phosphorylation resulted in an expected small rightward shift to 5.97, although statistical significance was not reached. Protein kinase C-induced phosphorylation also stimulated (80%) the maximal myofibrillar Mg2+-ATPase activity. [+]EMD 60263 (3 μM) produced a leftward shift of the myofibrillar pCa2+/Mg2+-ATPase curve which was unaffected by prior protein kinase C-induced phosphorylation. In conclusion, the findings with isolated myofibrils from myocardium of anaesthetized open-chest pigs indicate that protein kinase C might be involved in the mechanism of reduced Ca2+ responsiveness of myofilaments in stunned myocardium. However, at this stage no differences could be found between the maximal activity of the Ca2+-stimulated Mg2+ -ATPase, the pCa50 and the degree of phosphorylation of myofibrils isolated from stunned and non-stunned myocardium

Evaluating the Precision of Estimation of an Inequality Constrained Ridge Regression Estimator by the Bootstrap Methods

markdownabstractAbstract The effects of nisoldipine (0.1 μg/kg/min; n = 9) or its solvent (n = 9) were studied in pigs, in which left anterior descending coronary artery (LADCA) blood flow in both groups was reduced to 20% of baseline for 60 min and reperfused for 2 hr. Infusions were started at 30 min of ischaemia and lasted throughout reperfusion. In both groups, flow reduction abolished regional contractile function and caused similar decreases in the level of creatine phosphate (CP; by 70%) and the energy charge (from 0.91 to 0.69), mean arterial blood pressure (by 25%), LVdP/dtmax (by 30%) and cardiac output (by 30%). During ischaemia LADCA blood flow slightly increased (from 14 ± 8 to 24 ± 6mL/min/ 100 g; P < 0.05) in the nisoldipine-treated animals, resulting in an increase in CP to 91 ± 24% of baseline and preventing further decreases in energy charge, as observed in the solvent-treated animals. After 2 hr of reperfusion in neither group return of contractile function of the post-ischaemic myocardium was observed. Post-ischaemic blood flow in the nisoldipine-treated pigs increased from 24 ± 6 mL/min/100 g to 76 ± 14 mL/min/100 g and from 19 ± 6 mL/min/100 g to 41 ± 6 mL/min/100 g in the solvent-treated animals (P < 0.05) after 2 hr of reperfusion. Myocardial work was significantly higher in the nisoldipinetreated animals (111 ± 15 mmHg.L/min as compared to 69 ± 14 mmHg.L/min in the solvent-treated pigs after 2 hr of ischaemia). The energy charge of the post-ischaemic myocardium was similar for both groups (0.84 ± 0.02 for the nisoldipine-treated and 0.83 ± 0.03 for the solvent-treated animals). The rate of sarcoplasmic reticular Ca2+ uptake of the non-ischaemic segment of the nisoldipine-treated animals was 61% higher (P < 0.05) than that of the solvent-treated animals. In the post-ischaemic myocardium similar rates of Ca2+ uptake were found in both groups, but the activities were markedly lower as compared to the non-ischaemic myocardium. It is concluded that nisoldipine increases blood flow during reperfusion, which may have been caused by coronary vasodilatation. However, attenuation of the “no-reflow” phenomenon also contributed, since more rapid rephosphorylation of ADP leading to an increase in CP during ischaemia may have preserved jeopardized cells. Moreover, nisoldipine increases the sarcoplasmic reticular Ca2+ pump activity independent of ischaemia, which may have contributed in reducing the Ca2+ overload. Abbreviation

Uptake of triiodothyronine and triiodothyroacetic acid in neonatal rat cardiomyocytes: effects of metabolites and analogs

Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and [125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas the relative nuclear binding of the two tracers was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05<P<0.1). In contrast, 10 microM 3,5-T2, Triac, or tetraiodothyroacetic acid (Tetrac) did not affect T3 uptake after 15 min or after 24 h. Diiodothyropropionic acid (DITPA) (10 microM) reduced 15-min T3 uptake by about 24% (P<0.05), but it had a greater effect after 4 h (56%; P<0.001). Exposure to 10 nM DITPA during culture reduced cellular T3 uptake, as did 10 nM T3, suggesting down-regulation of the plasma membrane T3 transporters. We conclude that i) Triac is taken up by cardiomyocytes; ii) 3,3'-T2 and, to a lesser extent, DITPA and T4 interfere with plasma membrane transport of T3, whereas 3,5-T2, Triac, or Tetrac do not; iii) the transport mechanism for Triac is probably different from that for T3

In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics

The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSVβ-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods