Bertrand Crozatier

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to βadrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein–kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein–kinin system could be involved in the development or progression of cardiovascular diseases.

Altered balance between matrix gelatinases (MMP-2 and MMP-9) and their tissue inhibitors in human dilated cardiomyopathy: potential role of MMP-9 in myosin-heavy chain degradation.

End‐stage of human dilated cardiomyopathy (DCM) is characterized by myocyte loss and fibrosis, and associated with ventricular dilatation and reduced cardiac function. Matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) have been involved in the myocardial remodeling.

Protein Kinase C Isoform Expression in Normal and Failing Rabbit Hearts

Protein kinase C (PKC) is activated by alpha-adrenergic stimulation. Molecular analysis showed that PKC consists of a family of at least 12 isozymes. Studies of their distribution in the heart showed conflicting results. The first goal of our study was thus to characterize cardiac PKC in normal rabbits. PKC plays an important role in gene expression, cell growth, and differentiation and is involved in the hypertrophy phase of cardiac overload, but since its expression has never been evaluated in heart failure, the second goal of our study was to evaluate PKC activity and isoform expression in rabbits with heart failure induced by a double hemodynamic overload (aortic insufficiency followed by an aortic stenosis). In the first part of the study, PKC isoform expression analyzed in normal rabbits by immunoblotting showed that isoforms alpha, beta, epsilon, and zeta were expressed along with PKC gamma, which had never been detected in the heart. PKC gamma expression was also identified by polymerase chain reaction, and immunofluorescence techniques showed a localization on intercalated disks associated with the membrane localization observed with the other isoforms. In the second part of the study, PKC activity, content, and isoform expression showed a decrease of 37% in the failing group. PKC immunodetection with a monoclonal antibody (Mab 1.9) recognizing the catalytic domain of all PKC isoforms revealed a 20% decrease in the failing ventricles compared with normal left ventricles. Expressed PKC isoforms quantified by Western blot showed, in the failing heart group compared with the control group, a decrease of 27%, 32%, 16%, and 9% of PKC alpha, PKC beta 1, PKC gamma, and PKC epsilon, respectively, whereas PKC zeta was not significantly modified. These results show that, in heart failure, PKC activity and expression of Ca(2+)-dependent PKC isoforms are decreased. This may lead to alterations of PKC-induced phosphorylations.

Stimulation of Bradykinin B1 Receptors Induces Vasodilation in Conductance and Resistance Coronary Vessels in Conscious Dogs Comparison With B2 Receptor Stimulation

Background—Constitutive bradykinin B1 receptors have been identified in dogs; however, their physiological implications involving the coronary circulation remain to be determined. This study examined, in conscious dogs, the coronary response to des-Arg9-bradykinin (a B1 receptor agonist) and the mechanisms involved. Methods and Results—Eleven dogs were instrumented with a left ventricular micromanometer, a circumflex coronary catheter, a cuff occluder, a Doppler flow probe, and ultrasonic crystals to measure coronary blood flow velocity (CBFv) and coronary diameter (CD). Intracoronary des-Arg9-bradykinin (3 to 100 ng/kg) and bradykinin (0.1 to 10 ng/kg) did not modify systemic hemodynamics but dose-dependently increased CBFv and CD. Des-Arg9-bradykinin was less potent than bradykinin. Hoe 140 (a B2 antagonist, 10 μg/kg) abolished the effects of bradykinin but did not influence the effects of des-Arg9-bradykinin. When CBFv increase was prevented by the cuff occluder, CD responses to bradykinin and des-Arg9...

Stretch-induced modifications of myocardial performance: from ventricular function to cellular and molecular mechanisms

The purpose of this review is to give an integrative view of the effect of stretch on the myocardium from ventricular function changes to cellular and molecular mechanisms. The general approach will be to discuss the cellular and molecular events which can explain the findings obtained in the whole ventricle. Following the historical development, the classical analysis of the Starling mechanism and the basis of the length-dependent process are rapidly reviewed. We then analyze in greater detail the recent findings on the mechanisms of length-dependent activation changes in contractile protein affinity for calcium, changes in intracellular calcium release, action potential changes and stretch-activated ion channels and discuss the opposite effects of stretch on ventricular contraction (mainly deactivation shortening and viscoelastic properties of the myocardium). Besides the short-term contractile response to stretch, the longer-term effects of myocardial stretch which lead to ventricular hypertrophy will be also rapidly reviewed.

Epac activation induces histone deacetylase nuclear export via a Ras-dependent signalling pathway.

Epac (Exchange protein directly activated by cAMP) is a sensor for cAMP and represents a novel mechanism for governing cAMP signalling. Epac is a guanine nucleotide exchange factor (GEF) for the Ras family of small GTPases, Rap. Previous studies demonstrated that, in response to a prolonged beta-adrenergic stimulation Epac induced cardiac myocyte hypertrophy. The aim of our study was to further characterize Epac downstream effectors involved in cardiac myocyte growth. Here, we found that Epac led to the activation of the small G protein H-Ras in primary neonatal cardiac myocytes. A Rap GTPase activating protein (RapGAP) partially inhibited Epac-induced H-Ras activation. Interestingly, we found that H-Ras activation involved the GEF domain of Epac. However, Epac did not directly induce exchange activity on this small GTPase protein. Instead, the effect of Epac on H-Ras activation was dependent on a signalling cascade involving phospholipase C (PLC)/inositol 1,3,5 triphosphate receptor (IP3R) and an increase intracellular calcium. In addition, we found that Epac activation induced histone deacetylase type 4 (HDAC4) translocation. Whereas HDAC5 alone was unresponsive to Epac, it became responsive to Epac in the presence of HDAC4 in COS cells. Consistent with its effect on HDAC cytoplasmic shuttle, Epac activation also increased the prohypertrophic transcription factor MEF2 in a CaMKII dependent manner in primary cardiac myocytes. Thus, our data show that Epac activates a prohypertrophic signalling pathway which involves PLC, H-Ras, CaMKII and HDAC nuclear export.

PKC-delta and PKC-epsilon: foes of the same family or strangers?

Protein kinase C (PKC) is a family of 10 serine/threonine kinases divided into 3 subfamilies, classical, novel and atypical classes. Two PKC isozymes of the novel group, PKCε and PKCδ, have different and sometimes opposite effects. PKCε stimulates cell growth and differentiation while PKCδ is apoptotic. In the heart, they are among the most expressed PKC isozymes and they are opposed in the preconditioning process with a positive role of PKCε and an inhibiting role of PKCδ. The goal of this review is to analyze the structural differences of these 2 enzymes that may explain their different behaviors and properties.

Role of creatine kinase in cardiac excitation-contraction coupling: studies in creatine kinase-deficient mice

To understand the role of creatine kinase (CK) in cardiac excitation‐contraction coupling, CK‐deficient mice (CK–/–) were studied in vitro and in vivo. In skinned fibers, the kinetics of caffeine‐induced release of Ca2+ was markedly slowed in CK–/– mice with a partial restoration when glycolytic substrates were added. These abnormalities were almost compensated for at the cellular level: the responses of Ca2+ transient and cell shortening to an increased pacing rate from 1 Hz to 4 Hz were normal with a normal post‐rest potentiation of shortening. However, the post‐rest potentiation of the Ca2+ transient was absent and the cellular contractile response to isoprenaline was decreased in CK–/– mice. In vivo, echocardiographically determined cardiac function was normal at rest but the response to isoprenaline was blunted in CK–/– mice. Previously described compensatory pathways (glycolytic pathway and closer sarcoplasmic reticulum‐mitochondria interactions) allow a quasi‐normal SR function in isolated cells and a normal basal in vivo ventricular function, but are not sufficient to cope with a large and rapid increase in energy demand produced by β‐adrenergic stimulation. This shows the specific role of CK in excitation‐contraction coupling in cardiac muscle that cannot be compensated for by other pathways.

Preload-induced curvilinearity of left ventricular end-systolic pressure-volume relations. Effects on derived indexes in closed-chest dogs.

End-systolic pressure-volume relations (ESPVRs) were analyzed in 10 closed-chest autonomically blocked dogs before and after volume loading that restored end-diastolic volume to its value measured in the control conscious state. Dogs had been previously instrumented with a left ventricular pressure micromanometer and ultrasonic crystals for measurements of major, anteroposterior, and septum-free wall diameters. Left ventricular volume was calculated with an ellipsoidal model in the left ventricular cavity. ESPVRs obtained during caval occlusion after volume loading were curvilinear as shown by the division of the relation into two parts. The initial part of the relation had a significantly smaller ESPVR slope (Ees, 12.0 +/- 1.8 mm Hg/ml) and ESPVR volume-axis intercept (Vd, - 3.5 +/- 0.8 ml) than the final part of the relation (19.5 +/- 3.1 mm Hg/ml and 0.0 +/- 0.6 ml, respectively, p less than 0.01). The end-diastolic volume-peak dP/dt relation showed a similar curvilinearity when end-diastolic volumes were larger than 1.5-1.7 times the minimal end-diastolic volume reached during caval occlusion. ESPVRs were not different during aortic constriction and caval occlusion when end-diastolic volume was small. In contrast, with large end-diastolic volumes, Ees and Vd were significantly smaller during caval occlusion than during aortic constriction. The final part of ESPVR (with small end-diastolic volume) had the same slope and intercept as that during aortic constriction. We conclude that preload produces a curvilinearity of ESPVR that significantly modifies derived indexes when the range of preload changes is large.

Preserved Vasodilator Effect of Bradykinin in Dogs With Heart Failure

BACKGROUND In heart failure (HF), vasoconstrictor systems are activated and endothelium-derived vasodilation is blunted. Bradykinin, a potent vasodilator, may play an important role in this setting. However, it is not known whether its vasodilator effect is modified in HF. METHODS AND RESULTS Fourteen chronically instrumented dogs were studied in the control state and in pacing-induced HF (250 bpm for 3 weeks). The dose-dependent decrease in mean aortic pressure (MAP) induced by acetylcholine was significantly blunted in HF. In contrast, in both control and HF, bradykinin infusion caused similar dose-dependent decreases in MAP and increases in cardiac output (CO). This vasodilator effect of exogenous bradykinin was potentiated similarly in both states by enalaprilat, which blocks both angiotensin conversion and bradykinin degradation. For evaluating the role of endogenous bradykinin, the effects of enalaprilat were compared with those of ciprokiren, a pure renin inhibitor. In control, ciprokiren did not produce any effect. Enalaprilat, however, produced a significant decrease in MAP and a significant increase in CO, which were attributed to the inhibition of bradykinin degradation, because these effects were absent after pretreatment with Hoe 140 (a bradykinin B2 receptor antagonist). In contrast, in HF, vasodilator effects of ciprokiren were observed, but enalaprilat produced larger changes in MAP and CO, and after Hoe 140, the hemodynamic effects of enalaprilat were significantly decreased, showing the effects of endogenous bradykinin, which were similar to those measured in control. CONCLUSIONS In this model of HF with a blunted endothelium-derived vasodilation, the vasodilator effects of exogenous and endogenous bradykinin are preserved. These results suggest that bradykinin may play an important role in HF, in which vasoconstriction is present and endothelium-dependent vasodilation is blunted.