Gender issues in cardiovascular diseases. Focus on energy metabolism.

It is increasingly recognized that sex and gender differences (S&G) influence cardiovascular diseases (CVD), greatly impacting disease management. In terms of definition, sex refers to biological aspects, gender effects being mainly related to socio-cultural factors. Both sex and gender are interpenetrated in humans and difficult to separate. This is more clearly feasible in animal models where sex effects largely predominate. As alterations in energy metabolism are essential features of cardiovascular diseases, sexual dimorphism of energy metabolism and more specifically mitochondria occupies a place of choice. This review presents the basis of sex and gender differences in the cardiovascular pathophysiology, and how it mainly affects woman diseases, effectiveness of therapies and clinical outcome. These differences rely on complex molecular mechanisms that are still poorly understood because of the under-representation of females/women in experimental and clinical studies. Finally, the differing psychological and biological phases of woman's life are largely underestimated. This review presents an overview of the field with focus on differences in cardiac energy metabolism, which are illustrated with specific examples.

[Development and cardiomyopathy: serum response factor, a key protein]. / Développement et cardiomyopathie: le facteur de réponse au sérum, une protéine clé.

Serum response factor (SRF) is a widely expressed transcription factor involved in the transcription of various genes linked to muscle differentiation and cellular growth. Recent studies show the pivotal role of SRF in orchestrating genetic programs essential for cardiac development and function. Dominant negative isoforms of SRF resulting from caspase cleavage or alternative splicing have been identified in different forms of cardiomyopathies. This review summarizes the role of SRF, its structure, function and its role in human cardiopathies. Finally, we discuss the results of recently developed murine models which address the role of SRF in the adult heart in vivo. The existing biological data suggest that SRF could be a target of neurohumoral activation which is involved in myocardial hypertrophy. Conversely, inhibition of SRF activity in different murine models leads to dilated cardiomyopathy.

An overlapping CArG/octamer element is required for regulation of desmin gene transcription in arterial smooth muscle cells.

The desmin gene encodes an intermediate filament protein that is present in skeletal, cardiac, and smooth muscle cells. This study shows that the 4-kb upstream region of the murine desmin promoter directs expression of a lacZ reporter gene throughout the heart from E7.5 and in skeletal muscle and vascular smooth muscle cells from E9. 5. The distal fragment (-4005/-2495) is active in arterial smooth muscle cells but not in venous smooth muscle cells or in the heart in vivo. It contains a CArG/octamer overlapping element (designated CArG4) that can bind the serum response factor (SRF) and an Oct-like factor. The desmin distal fragment can replace a SM22alpha regulatory region (-445/-126) that contains two CArG boxes, to cis-activate a minimal (-125/+65) SM22alpha promoter fragment in arterial smooth muscle cells of transgenic embryos. lacZ expression was abolished when mutations were introduced into the desmin CArG4 element that abolished the binding of SRF and/or Oct-like factor. These data suggest that a new type of combined CArG/octamer element plays a prominent role in the regulation of the desmin gene in arterial smooth muscle cells, and SRF and Oct-like factor could cooperate to drive specific expression in these cells.

Transplacental injection of somite-derived cells in mdx mouse embryos for the correction of dystrophin deficiency.

Duchenne muscular dystrophy (DMD) is a lethal recessive disease caused by the absence of dystrophin in skeletal muscle, heart and other tissues. No cure is available at present for DMD. Here we describe a new strategy for the correction of dystrophin deficiency based on the transplantation of normal somite-derived cells into mdx mouse embryos. Somite-derived cells were isolated from E11.5 transgenic mouse embryos expressing the LacZ gene under the control of the muscle-specific desmin promoter and injected into the uterine circulation of pregnant mdx mice at gestational days E11.5-E17. Approximately 30% of the injected mdx embryos survived the procedure. Donor somite-derived cells were able to cross the placenta and migrate into host embryonic tissues. The pattern of donor cell distribution in host tissues depended on the gestational age of the transplanted embryos. Cells were found in hindlimb muscles, diaphragm, heart and ribs in E11.5 treated embryos and in the skull, ribs, vertebrae and lung of E15-E17 treated embryos. Normal dystrophin transcripts were detected in muscle and bone by RT-PCR. Histochemical analysis showed co-localization of LacZ and dystrophin expression in 5% of soleus and quadriceps muscle fibres and in 4% of heart myocytes of two of seven 8-week-old treated mdx mice.

A crucial role for Pax3 in the development of the hypaxial musculature and the long-range migration of muscle precursors.

Activated by dorsalizing and lateralizing signals, the Pax3 gene is an early marker for the entire paraxial mesoderm and its dorsal derivative, the dermomyotome. Later, its expression becomes restricted to the lateral dermomyotome and to the migratory muscle precursors giving rise to the hypaxial musculature. To understand better the role that Pax3 plays during development of paraxial mesoderm-derived structures, we followed the development of the musculature and skeleton in the murine Pax3 mutant Splotch. We found that the mutant dermomyotomes and myotomes failed to organize and to elongate medially and laterally, leading to the reduction and malformation of the entire trunk musculature. Mutants lacked ventral aspects of the body wall musculature and muscles derived from migratory myoblasts, suggesting a crucial function for Pax3 in the long-range migration of muscle precursors giving rise to the ventral hypaxial musculature. In addition, severe malformations were detected in the skeleton. The axial and appendicular skeleton displayed malformations and in particular multiple bone fusions.

Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton.

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).

Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle.

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des -/-) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362-366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255- 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des -/- mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.

Null mutation in the desmin gene gives rise to a cardiomyopathy.

A null mutation in the desmin gene has been introduced into the germ line of mice. Such mice develop and reproduce normally proving that desmin is not needed either for the formation of the heart or the alignment of functioning myofibrils. However, cardiovascular lesions and a skeletal myopathy were observed in growing and adult mice. In the present study we have carried out a detailed analysis of these cardiac lesions. Homozygous mutant mice, which were confirmed to lack expression of desmin mRNA and desmin protein in the heart, were revealed by electron microscopy to contain degenerating cardiomyocytes as early as 5 days post-partum. At 10 days post-partum and onwards the degeneration of cardiomyocytes gave rise to areas with an accumulation of macrophages, fibrosis and calcification preferentially in the inter-ventricular septum and the free wall of the right ventricle. The localization of the lesions mainly to these sites suggested that it is not the work load and contractions per se which were the pathogenic events leading to the cardiomyopathy. It might be that stress related to lengthening of the myocytes occur more in the right ventricle than in the left. At the ultrastructural level changes in the intercalated discs, disruption of the sarcolemma and supercontraction of myofibrils seemed to be the key events leading to cardiomyocyte death. Thus, the intermediate filaments are required to maintain the basic integrity of cardiomyocytes and especially the link between the intermediate filaments and the sarcolemma appear more important than previously realized.

The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies.

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.

N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA.

Human lactoferrin (hLF), a protein involved in host defence against infection and excessive inflammation, interacts with heparin, the lipid A moiety of bacterial lipopolysaccharide, human lysozyme (hLZ) and DNA. To determine which region of the molecule is important in these interactions, solid-phase ligand binding assays were performed with hLF from human milk (natural hLF) and N-terminally deleted hLF variants. Iron-saturated and natural hLF bound equally well to heparin, lipid A, hLZ and DNA. Natural hLF lacking the first two N-terminal amino acids (Gly1-Arg2) showed reactivities of one-half, two-thirds, one-third and one-third towards heparin, lipid A, hLZ and DNA respectively compared with N-terminally intact hLF. A lack of the first three residues (Gly1-Arg2-Arg3) decreased binding to the same ligands to one-eighth, one-quarter, one-twentieth and one-seventeenth respectively. No binding occurred with a mutant lacking the first five residues (Gly1-Arg2-Arg3-Arg4-Arg5). An anti-hLF monoclonal antibody (E11) that reacts to an N-lobe epitope including Arg5 completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg2-Arg3-Arg4-Arg5, has a decisive role in the interaction of hLF with heparin, lipid A, hLZ and DNA. The role of limited N-terminal proteolysis of hLF in its anti-infective and anti-inflammatory properties is discussed.

The SM 22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice.

The transcriptional signals underlying smooth muscle differentiation are currently unknown. We report here the complete sequence and characterization of the single mouse gene for the smooth muscle-specific protein SM 22 and the transcriptional activity of its promoter in cultured smooth muscle cells in vitro and in transgenic mice. In the transgenic animals, promoter constructs ranging in length from 445 to 2126 bp directed reporter expression initially in the heart and the somites of embryos and subsequently in the arteries of the vascular system, but in none of the visceral muscles, nor in the veins. Expression in the heart was spatially restricted to the presumptive right ventricle and outflow tract and disappeared in the adult. Likewise, expression in the somites was only transitory and was not observed after about 14.5 days post coitum in the embryo. In the adult mouse, SM 22 promoter activity persisted in the smooth muscle cells of the arteries and was still notably absent from other smooth muscles, despite the ubiquitous presence of the endogenous SM 22 protein. These findings on the transcriptional activity of a smooth muscle promoter in vivo reveal the existence of different differentiation programmes for smooth muscle cells in the veins and the arteries and raise the expectation of a further subdivision of programmes among the visceral muscles.

Cardiovascular lesions and skeletal myopathy in mice lacking desmin.

In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues.