George D. Giraud

Hemodynamic changes in pregnancy

The basic mechanisms that underlie alterations in the physiology of pregnancy are virtually unknown. Basal oxygen consumption increases by some 50 mL/min in pregnant women at term. Blood volume increases gradually over gestation as does red cell mass. Cardiac output increases by some 50% by mid-third trimester. Stroke volume and heart rate increase over the course of pregnancy with heart rate increasing gradually until term. The heart of the pregnant woman remodels dramatically in the first few weeks of pregnancy; end diastolic volume increases. Stroke volume is augmented by the increase in end diastolic volume and maintenance of ejection fraction through a possible increase in contractile force. Systolic and diastolic blood pressures drop during normal pregnancy. There is evidence of blood vessel remodeling in all vessels. Venous compliance and venous blood volume are increased. Renal plasma flow increases by some 70% in pregnancy with glomerular filtration rate increasing by 50% by unknown mechanisms. The complex hormonal environment is changing throughout pregnancy. In summary, under the influence of circulating chemical mediators blood flow is redistributed to the uterus, breast, and kidney.

Angiotensin II stimulates hyperplasia but not hypertrophy in immature ovine cardiomyocytes

Rat and sheep cardiac myocytes become binucleate as they complete the ‘terminal differentiation’ process soon after birth and are not able to divide thereafter. Angiotensin II (Ang II) is known to stimulate hypertrophic changes in rodent cardiomyocytes under both in vivo and in vitro conditions via the AT1 receptor and intracellular extracellular regulated kinase (ERK) signalling cascade. We sought to develop culture methods for immature sheep cardiomyocytes in order to test the hypothesis that Ang II is a hypertrophic agent in the immature myocardium of the sheep. We isolated fetal sheep cardiomyocytes and cultured them for 96 h, added Ang II and phenylephrine (PE) for 48 h, and measured footprint area and proliferation (5‐bromo‐2′‐deoxyuridine (BrdU) uptake) separately in mono‐ vs. binucleate myocytes. We found that neither Ang II nor PE changed the footprint area of mononucleated cells. PE stimulated an increase in footprint area of binucleate cells but Ang II did not. Ang II increased myocyte BrdU uptake compared to serum free conditions, but PE did not affect BrdU uptake. The MAP kinase kinase (MEK) inhibitor UO126 prevented BrdU uptake in Ang II‐stimulated cells and prevented cell hypertrophy in PE‐stimulated cells. This paper establishes culture methods for immature sheep cardiomyocytes and reports that: (1) Ang II is not a hypertrophic agent; (2) Ang II stimulates hyperplastic growth among mononucleate myocytes; (3) PE is a hypertrophic agent in binucleate myocytes; and (4) the ERK cascade is required for the proliferation effect of Ang II and the hypertrophic effect of PE.

Placental insufficiency decreases cell cycle activity and terminal maturation in fetal sheep cardiomyocytes.

Umbilicoplacental embolization (UPE) in sheep has been used to investigate the effects of placental insufficiency on fetal development. However, its specific effects on the heart have been little studied. The aim of this study was to determine the effects of placental insufficiency, induced by UPE, on cardiomyocyte size, maturation and proliferation. Instrumented fetal sheep underwent UPE for either 10 or 20 days. Hearts were collected at 125 ± 1 days (10 day group) or 136 ± 1 days (20 day group) of gestation (term ∼145 days). Cell size, maturational state (as measured by the proportion of binucleated myocytes) and cell cycle activity (as measured by positive staining of cells for Ki‐67) were determined in dissociated cardiomyocytes. UPE fetuses were hypoxaemic, but mean arterial pressures were not different from controls. UPE fetuses were lighter than control fetuses (10 days: −21%, P < 0.05; 20 days: −27%, P < 0.01) and had smaller hearts, but heart weight was appropriate for body weight. Neither lengths nor widths were different between control and UPE cardiomyocytes at either age. Ten days of UPE did not significantly alter the proportion of binucleated myocytes or cell cycle activity in either ventricle. However, 20 days of UPE reduced cell cycle activity in both ventricles by ∼70% (P < 0.05); the proportion of binucleated myocytes was also lower in UPE fetuses at this age (left ventricle: 31.1 ± 12.0 versus 46.0 ± 6.6%, P < 0.05; right ventricle: 29.4 ± 12.3 versus 46.3 ± 5.3%, P < 0.05). It is concluded that in the absence of fetal arterial hypertension, placental insufficiency is associated with substantially depressed growth of the heart through suppressed proliferation and maturation of cardiomyocytes.

Thyroid hormone drives fetal cardiomyocyte maturation

Tri‐iodo‐≤sc≥l≤/sc≥‐thyronine (T3) suppresses the proliferation of near‐term serum‐stimulated fetal ovine cardiomyocytes in vitro. Thus, we hypothesized that T3 is a major stimulant of cardiomyocyte maturation in vivo. We studied 3 groups of sheep fetuses on gestational days 125‐130 (term ~145 d): a T3‐infusion group, to mimic fetal term levels (plasma T3 levels increased from ~0.1 to ~1.0 ng/ml; t½~24 h); a thyroidectomized group, to produce low thyroid hormone levels; and a vehicle‐infusion group, to serve as intact controls. At 130 d of gestation, sections of left ventricular freewall were harvested, and the remaining myocardium was enzymatically dissociated. Proteins involved in cell cycle regulation (p21, cyclin D1), proliferation (ERK), and hypertrophy (mTOR) were measured in left ventricular tissue. Evidence that elevated T3 augmented the maturation rate of cardiomyocytes included 14% increased width, 31% increase in binucleation, 39% reduction in proliferation, 150% reduction in cyclin D1 protein, and 500% increase in p21 protein. Increased expression of phospho‐mTOR, ANP, and SERCA2a also suggests that T3 promotes maturation and hypertrophy of fetal cardiomyocytes. Thyroidectomized fetuses had reduced cell cycle activity and binucleation. These findings support the hypothesis that T3 is a prime driver of prenatal cardiomyocyte maturation.—Chattergoon, N. N., Giraud, G. D., Louey, S., Stork, P., Fowden, A. L., Thornburg, K. L. Thyroid hormone drives fetal cardiomyocyte maturation FASEB J. 26, 397–408 (2012). www.fasebj.org

Regulation of the Cardiomyocyte Population in the Developing Heart

During fetal life the myocardium expands through replication of cardiomyocytes. In sheep, cardiomyocytes begin the process of becoming terminally differentiated at about 100 gestation days out of 145 days term. In this final step of development, cardiomyocytes become binucleated and stop dividing. The number of cells at birth is important in determining the number of cardiomyocytes for life. Therefore, the regulation of cardiomyocyte growth in the womb is critical to long term disease outcome. Growth factors that stimulate proliferation of fetal cardiomyocytes include angiotensin II, cortisol and insulin-like growth factor-1. Increased ventricular wall stress leads to short term increases in proliferation but longer-term loss of cardiomyocyte generative capacity. Two normally circulating hormones have been identified that suppress proliferation: atrial natriuretic peptide (ANP) and tri-iodo-L-thyronine (T₃). Atrial natriuretic peptide signals through the NPRA receptor that serves as a guanylate cyclase and signals through cGMP. ANP powerfully suppresses mitotic activity in cardiomyocytes in the presence of angiotensin II in culture. Addition of a cGMP analog has the same effect as ANP. ANP suppresses both the extracellular receptor kinases and the phosphoinositol-3 kinase pathways. T₃ also suppresses increased mitotic activity of stimulated cardiomyocytes but does so by increasing the cell cycle suppressant, p21, and decreasing the cell cycle activator, cyclin D1.

Effects of estrogen and progestin on aortic size and compliance in postmenopausal women

OBJECTIVE Our purpose was to determine whether sex steroids alter aortic size and compliance in postmenopausal women. STUDY DESIGN Twenty-six postmenopausal women were randomized to receive either conjugated estrogens 0.625 mg per day (group 1) or conjugated estrogens 0.625 mg per day and medroxyprogesterone 2.5 mg per day (group 2). Aortic cross-sectional area was measured by magnetic resonance imaging before and after 3 months of hormone therapy. RESULTS Estradiol levels increased in both group 1 and group 2 (p < 0.0001). Ascending aortic cross-sectional area increased from 439 +/- 7 mm2 to 466 +/- 7 mm2 in group 1 (p < 0.008) but was unchanged in group 2. Within the range of aortic pressures studied, no change in aortic compliance could be detected. CONCLUSION Estrogen therapy in postmenopausal women was associated with an increase in aortic size; but this effect was not detectable with the addition of progestin. The potential antagonistic effect of progestin on estrogen-induced aortic enlargement suggests that the favorable cardiovascular effects of postmenopausal estrogen therapy cannot be automatically extended to the combination estrogen-progestin.

Heterogeneous ventricular sympathetic innervation, altered β-adrenergic receptor expression, and rhythm instability in mice lacking the p75 neurotrophin receptor

Sympathetic nerves stimulate cardiac function through the release of norepinephrine and the activation of cardiac beta(1)-adrenergic receptors. The sympathetic innervation of the heart is sculpted during development by chemoattractive factors including nerve growth factor (NGF) and the chemorepulsive factor semaphorin 3a. NGF acts through the TrkA receptor and the p75 neurotrophin receptor (p75(NTR)) in sympathetic neurons. NGF stimulates sympathetic axon extension into the heart through TrkA, but p75(NTR) modulates multiple coreceptors that can either stimulate or inhibit axon outgrowth. In mice lacking p75(NTR), the sympathetic innervation density in target tissues ranges from denervation to hyperinnervation. Recent studies have revealed significant changes in the sympathetic innervation density of p75NTR-deficient (p75(NTR-/-)) atria between early postnatal development and adulthood. We examined the innervation of adult p75(NTR-/-) ventricles and discovered that the subendocardium of the p75(NTR-/-) left ventricle was essentially devoid of sympathetic nerve fibers, whereas the innervation density of the subepicardium was normal. This phenotype is similar to that seen in mice overexpressing semaphorin 3a, and we found that sympathetic axons lacking p75(NTR) are more sensitive to semaphorin 3a in vitro than control neurons. The lack of subendocardial innervation was associated with decreased dP/dt, altered cardiac beta(1)-adrenergic receptor expression and sensitivity, and a significant increase in spontaneous ventricular arrhythmias. The lack of p75(NTR) also resulted in increased tyrosine hydroxylase content in cardiac sympathetic neurons and elevated norepinephrine in the right ventricle, where innervation density was normal.

The effects of anaemia as a programming agent in the fetal heart.

The intrauterine environment plays a powerful role in determining the life‐long risk of cardiovascular disease. A number of stressors are well known to affect the development of the cardiovascular system in utero including over/under maternal nutrition, excess glucocorticoid and chronic hypoxia. Chronic fetal anaemia in sheep is a complex stressor that alters cardiac loading conditions, causes hypoxic stress and stimulates large changes in flow to specific tissues, including large increases in resting coronary blood flow and conductance. Decreased viscosity can account for approximately half of the increased flow. It appears that immature hearts are ‘plastic’ in that increases in coronary conductance with fetal anaemia persist into adulthood even if the anaemia is corrected before birth. These large changes in conductance are possible only through extensive remodelling of the coronary tree. Adult hearts that were once anaemic in utero are more resistant to hypoxic stress as adults but it is not known whether such an adaptation would be deleterious in later life. These studies indicate the need for investigation into the basic mechanisms of coronary tree remodelling in the immature myocardium. New information on these mechanisms is likely to lead to better prevention of and therapies for adult‐onset coronary disease.

Spontaneous echo contrast and hemorheologic abnormalities in cerebrovascular disease.

Background and Purpose Spontaneous echo contrast (SEC) is thought to represent a risk factor for cardioembolic stroke. In vitro studies suggest that SEC results from interaction between red cells and fibrinogen. To better understand the relation between SEC and stroke and to investigate the in vivo genesis of SEC, we examined the relation between SEC, the constituents of the blood, and plasma and serum viscosity in patients with acute stroke or chronic cerebrovascular disease. Methods Fifty patients with acute stroke or chronic cerebrovascular disease referred for transesophageal echocardiogram (TEE) were studied by transthoracic echocardiography and TEE. Complete blood count, fibrinogen, albumin, γ-globulin, and plasma and serum viscosity determinations were made. Left atrial SEC was graded as absent, mild, or marked by means of TEE. Results SEC was absent in 31 patients, mild in 10 patients, and marked in 9 patients. Higher grade of SEC was associated with a significantly greater percentage of patients with atrial fibrillation and larger left atrial dimension. Atrial fibrillation was present in 23% of the patients in the SEC absent group, 50% of the patients in the mild SEC group, and 78% of the patients in the marked SEC group (P<.01). Left atrial diameter averaged 3.8±0.6 cm in the SEC absent group, 4.3±1.1 in the mild SEC group, and 4.9±0.7 in the marked SEC group (P<.001). Hematocrit, white blood cell count, and platelet count did not differ among the three groups. Fibrinogen, γ-globulin, plasma viscosity, and serum viscosity values were all significantly higher in the presence of SEC (P<.05). Fibrinogen values were 361 ±97 mg/dL in the SEC absent group and 427±135 mg/dL in the marked SEC group. γ-Globulin levels were 0.75±0.23 g/dL in the SEC absent group and 1.06±0.48 g/dL in the marked SEC group. Both plasma viscosity (1.97 cp) and serum viscosity (1.64 cp) were higher in the marked SEC group than in the SEC absent group (1.77 and 1.50 cp, respectively). Conclusions In patients with acute stroke or chronic cerebrovascular disease, the severity of SEC was not related to albumin, hematocrit, white cell count, or platelet count but rather to elevated fibrinogen levels and concomitant increases in both plasma and serum viscosity. Moreover, increasing grade of SEC was associated with significantly increased left atrial diameter and a higher percentage of patients in atrial fibrillation.

Atrial natriuretic peptide inhibits angiotensin II-stimulated proliferation in fetal cardiomyocytes

The role of atrial natriuretic peptide (ANP) in regulating fetal cardiac growth is poorly understood. Angiotensin II (Ang II) stimulates proliferation in fetal sheep cardiomyocytes when growth is dependent on the activity of the mitogen‐activated protein kinase (MAPK) and phosphoinositol‐3‐kinase (PI3K) pathways. We hypothesized that ANP would suppress near‐term fetal cardiomyocyte proliferation in vitro and inhibit both the MAPK and PI3K pathways. Forty‐eight hour 5‐bromodeoxyuridine (BrdU) uptake (used as an index of proliferation) was measured in cardiomyocytes isolated from fetal sheep (135 day gestational age) in response to 100 nm Ang II with or without ANP (0.003–100 nm) or 1 μm 8‐bromo‐cGMP. The effects of these compounds on the MAPK and PI3K pathways were assessed by measuring extracellular signal‐regulated kinase (ERK) and AKT phosphorylation following 10 min of treatment with Ang II, ANP or 8‐bromo‐cGMP. In right ventricular myocytes (RV), the lowest dose of ANP (0.003 nm) inhibited Ang II‐stimulated BrdU uptake by 68%. Similarly, 8‐bromo‐cGMP suppressed Ang II‐stimulated proliferation by 62%. The same effects were observed in left ventricular (LV) cardiomyoytes but the RV was more sensitive to the inhibitory effects of ANP than the LV (P < 0.0001). Intracellular cGMP was increased by 4‐fold in the presence of 100 nm ANP. Ang II‐stimulated ERK and Akt phosphorylation was inhibited by 100 nm ANP. The activity of ANP may in part be cGMP dependent, as 8‐bromo‐cGMP had similar effects on the cardiomyocytes.