Dimitrios Georgakopoulos

The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy

Stress-induced mitogen-activated protein kinase (MAP) p38 is activated in various forms of heart failure, yet its effects on the intact heart remain to be established. Targeted activation of p38 MAP kinase in ventricular myocytes was achieved in vivo by using a gene-switch transgenic strategy with activated mutants of upstream kinases MKK3bE and MKK6bE. Transgene expression resulted in significant induction of p38 kinase activity and premature death at 7–9 weeks. Both groups of transgenic hearts exhibited marked interstitial fibrosis and expression of fetal marker genes characteristic of cardiac failure, but no significant hypertrophy at the organ level. Echocardiographic and pressure-volume analyses revealed a similar extent of systolic contractile depression and restrictive diastolic abnormalities related to markedly increased passive chamber stiffness. However, MKK3bE-expressing hearts had increased end-systolic chamber volumes and a thinned ventricular wall, associated with heterogeneous myocyte atrophy, whereas MKK6bE hearts had reduced end-diastolic ventricular cavity size, a modest increase in myocyte size, and no significant myocyte atrophy. These data provide in vivo evidence for a negative inotropic and restrictive diastolic effect from p38 MAP kinase activation in ventricular myocytes and reveal specific roles of p38 pathway in the development of ventricular end-systolic remodeling.

In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry

The mouse is the species of choice for creating genetically engineered models of human disease. To study detailed systolic and diastolic left ventricular (LV) chamber mechanics in mice in vivo, we developed a miniaturized conductance-manometer system. alpha-Chloralose-urethan-anesthetized animals were instrumented with a two-electrode pressure-volume catheter advanced via the LV apex to the aortic root. Custom electronics provided time-varying conductances related to cavity volume. Baseline hemodynamics were similar to values in conscious animals: 634 +/- 14 beats/min, 112 +/- 4 mmHg, 5.3 +/- 0.8 mmHg, and 11,777 +/- 732 mmHg/s for heart rate, end-systolic and end-diastolic pressures, and maximum first derivative of ventricular pressure with respect to time (dP/dtmax), respectively. Catheter stroke volume during preload reduction by inferior vena caval occlusion correlated with that by ultrasound aortic flow probe (r2 = 0.98). This maneuver yielded end-systolic elastances of 79 +/- 21 mmHg/microliter, preload-recruitable stroke work of 82 +/- 5.6 mmHg, and slope of dP/dtmax-end-diastolic volume relation of 699 +/- 100 mmHg.s-1.microliter-1, and these relations varied predictably with acute inotropic interventions. The control normalized time-varying elastance curve was similar to human data, further supporting comparable chamber mechanics between species. This novel approach should greatly help assess cardiovascular function in the blood-perfused murine heart.The mouse is the species of choice for creating genetically engineered models of human disease. To study detailed systolic and diastolic left ventricular (LV) chamber mechanics in mice in vivo, we developed a miniaturized conductance-manometer system. α-Chloralose-urethan-anesthetized animals were instrumented with a two-electrode pressure-volume catheter advanced via the LV apex to the aortic root. Custom electronics provided time-varying conductances related to cavity volume. Baseline hemodynamics were similar to values in conscious animals: 634 ± 14 beats/min, 112 ± 4 mmHg, 5.3 ± 0.8 mmHg, and 11,777 ± 732 mmHg/s for heart rate, end-systolic and end-diastolic pressures, and maximum first derivative of ventricular pressure with respect to time (dP/d t max), respectively. Catheter stroke volume during preload reduction by inferior vena caval occlusion correlated with that by ultrasound aortic flow probe ( r 2 = 0.98). This maneuver yielded end-systolic elastances of 79 ± 21 mmHg/μl, preload-recruitable stroke work of 82 ± 5.6 mmHg, and slope of dP/d t max-end-diastolic volume relation of 699 ± 100 mmHg ⋅ s-1 ⋅ μl-1, and these relations varied predictably with acute inotropic interventions. The control normalized time-varying elastance curve was similar to human data, further supporting comparable chamber mechanics between species. This novel approach should greatly help assess cardiovascular function in the blood-perfused murine heart.

Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Murine Cardiac Function A Cautionary Tail

The capacity to selectively mutate genes or create excessive or deleted gene expression in mice has yielded a powerful new approach to structure-function studies of cardiac proteins and their role in heart disease.1 As it happened, the molecular techniques required to generate such animals developed more rapidly than did the methods for studying the chamber physiological phenotype. Mainly because of these methodological limitations, studies to date have often presented hemodynamic data that would fail the standards usually applied in larger species. In particular, heart rates (HRs) and basal levels of systolic contraction are frequently depressed to a substantial degree. It has required a major leap of faith to assume that the physiological differences measured between genetically modified and wild-type animals under such conditions translate to the healthy heart or intact animal. Furthermore, in the understandable rush to assess the impact of molecular manipulations, the careful assessment of normal murine cardiac physiology has been left shortchanged. Only now are we beginning to see the results of such analysis, with evidence growing to support potentially important differences between mice and other mammalian species.2 Such data may be very important for properly interpreting the physiological consequences of targeted genetic manipulations, as reviewed by James et al3 in this issue of Circulation Research .nnAs with larger animals, compromises can and often must be made to balance the need for adequate control to precisely assess cardiac mechanics and physiological intactness to maintain near-normal physiology. Although conscious animal data are often considered the gold standard in larger species, they are not necessarily required to provide relevant or valuable hemodynamic insights. However, awareness of more intact normal physiology has always been critical to properly interpret data obtained in more invasive or isolated heart studies. Rarely has the discrepancy between conscious animal and anesthetized …

The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation.

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder resulting from mutations in genes encoding sarcomeric proteins. This typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. To better understand the disease pathogenesis, early (primary) abnormalities must be distinguished from evolving responses to the genetic defect. We did in vivo analysis using a mouse model of FHC with an Arg403Gln α-cardiac myosin heavy chain missense mutation, and used newly developed methods for assessing in situ pressure–volume relations. Hearts of young mutant mice (6 weeks old), which show no chamber morphologic or gross histologic abnormalities, had altered contraction kinetics, with considerably delayed pressure relaxation and chamber filling, yet accelerated systolic pressure rise. Older mutant mice (20 weeks old), which develop fiber disarray and fibrosis, had diastolic and systolic kinetic changes similar to if not slightly less than those of younger mice. However, the hearts of older mutant mice also showed hyperdynamic contraction, with increased end-systolic chamber stiffness, outflow tract pressure gradients and a lower cardiac index due to reduced chamber filling; all hallmarks of human disease. These data provide new insights into the temporal evolution of FHC. Such data may help direct new therapeutic strategies to diminish disease progression.

Minimal force‐frequency modulation of inotropy and relaxation of in situ murine heart

1 The normal influence of heart rate (HR) on cardiac contraction and relaxation in the mouse remains uncertain despite its importance in interpreting many genetically engineered models. Prior in vivo data have repeatedly shown positive effects only at subphysiological heart rates, yet depressed basal conditions and use of load‐dependent parameters probably have an impact on these results. 2 Open‐chest mice of various strains (n = 16, etomidate/urethane anaesthesia) were instrumented with a miniaturized pressure‐volume catheter employing absolute left ventricular (LV) volume calibration. HR was slowed (< 400 beats min−1) using ULFS‐49, and atrial or ventricular pacing was achieved via an intra‐oesophageal catheter. Pressure‐volume data yielded cardiac‐specific contractile indexes minimally altered by vascular load. 3 At a resting HR of 600 beats min−1, peak pressure‐rise rate (dP/dtmax) was 16 871 ± 2941 mmHg s−1 (mean ±s.d.) and the relaxation time constant was 3.9 ± 0.8 ms, similar to values in conscious animals. Within the broad physiological range (500‐850 beats min−1), load‐insensitive contractile indexes and relaxation rate varied minimally, whereas dP/dtmax peaked at 600 ± 25 beats min−1 and decreased at higher rates due to preload sensitivity. Contraction and relaxation were enhanced modestly (13‐15 %) at HRs of between 400 and 500 beats min−1. 4 The minimal force‐frequency dependence was explained by rapid calcium cycling kinetics, with a mechanical restitution time constant of 9 ± 2.7 ms, and by dominant sarcoplasmic reticular buffering (recirculation fraction of 93 ± 1 %). 5 The mouse normally has a very limited force‐frequency reserve at physiological HRs, unlike larger mammals and man. This is important to consider when studying disease evolution and survival of genetic models that alter calcium homeostasis and SR function.

β3-adrenoceptor deficiency blocks nitric oxide–dependent inhibition of myocardial contractility

The cardiac beta-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. beta-Adrenergic activation also increases cardiac nitric oxide (NO) production, which attenuates positive inotropy, suggesting a possible negative feedback mechanism. Recently, in vitro studies suggest that stimulation of the beta(3)-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous beta(3)-adrenoceptor deletion mutations, we tested the hypothesis that the beta(3)-adrenoceptor is responsible for beta-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, beta-adrenergic-stimulated inotropy was increased in beta(3)(-/-) mice, and similar hyper-responsiveness was seen in mice lacking endothelial NO synthase (NOS3). NOS inhibition augmented isoproterenol-stimulated inotropy in wild-type (WT), but not in beta(3)(-/-) mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not beta(3)(-/-), mice. NOS3 protein abundance was not changed in beta(3)(-/-) mice, and cardiac beta(3)-adrenoceptor mRNA was detected in both NOS3(-/-) and WT mice. These findings indicate that the beta(3)-adrenergic subtype participates in NO-mediated negative feedback over beta-adrenergic stimulation.

Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice.

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.

p38 MAP Kinase Mediates Inflammatory Cytokine Induction in Cardiomyocytes and Extracellular Matrix Remodeling in Heart

Background—Increasing evidence suggests that development of heart failure involves activation of stress-response inflammatory cytokines, including tumor necrosis factor-&agr; and interleukin-6. Yet, the myocyte contribution to their induction in failing hearts and the underlying regulatory mechanism in stressed myocardium remain unclear. Methods and Results—In cultured cardiac myocytes, specific activation of stress-activated mitogen-activated protein kinase, p38, by upstream activator MKK6bE led to significant induction of tumor necrosis factor-&agr; and interleukin-6 secretion, whereas treating cells with a selective p38 inhibitor (SB239068) significantly blocked the cytokine secretion from myocytes and increased their intracellular accumulation. Targeted expression of MKK6bE in transgenic hearts also resulted in a marked elevation in plasma tumor necrosis factor-&agr; and interleukin-6; oral administration of SB239068 resulted in a significant reduction in their plasma levels but an increase in intracardiac accumulation of both cytokines. MKK6bE transgenic hearts developed marked interstitial fibrosis with increased matrix metalloproteinase abundance and selective induction of tissue inhibitor of matrix metalloproteinase-1; this extracellular matrix remodeling was also significantly attenuated by p38 inhibition. Along with cytokine induction and extracellular remodeling, MKK6bE transgenic animals displayed impaired hemodynamic function, whereas p38 inhibition improved the cardiac performance and prolonged the survival of the animals. Conclusions—Stress-activated p38 kinase is a critical regulator of inflammatory response in cardiomyocytes with significant contribution to pathological remodeling in stressed myocardium. Inhibition of p38 may represent a useful therapeutic avenue to ameliorate cardiac pathology and heart failure evolution.

Angiogenesis in the Mouse Lung

When pulmonary arterial blood flow is obstructed in all mammals studied, there is a compensatory growth of the bronchial vasculature. This angiogenesis normally occurs through a proliferation of the systemic circulation to the intraparenchymal airways. It is an important pathophysiological process, not only in pulmonary vascular disease, but also in lung cancer, because the blood flow that supplies primary lung tumors arises from the systemic circulation. In the mouse, however, the systemic blood vessels that supply the trachea and mainstem bronchi do not penetrate into the intraparenchymal airways, as they do in all other larger species. In this study, we attempted to generate a new functional bronchial circulation in the mouse by permanently obstructing 40% of the pulmonary circulation. We quantified the systemic blood flow to the lung with fluorescent microspheres for 3 months after left pulmonary artery ligation. Results demonstrated that a substantial systemic blood flow to the lung that can eventually supply up to 15% of the normal pulmonary flow can be generated beginning 5-6 days after ligation. These new angiogenic vessels do not arise from the extraparenchymal bronchial circulation. Rather they enter the lung directly via a totally new vasculature that develops between the visceral and parietal pleuras, supplied by several intercostal arteries. This unique model of angiogenesis occurs in the absence of any hypoxic stimulus and mimics the vascular source of many lung tumors.