Danièle Charlemagne

Senescent Heart Compared With Pressure Overload–Induced Hypertrophy

Although systolic left ventricular (LV) function is normal in the elderly, aging is associated in rat papillary muscle with mechanical and sarcoplasmic reticulum Ca2+ ATPase alterations similar to those observed in the hypertrophied heart. However, alterations in the other calcium-regulating proteins implicated in contraction and relaxation are still unknown. To investigate alterations in LV function and calcium-regulating proteins, we measured hemodynamics and Na(+)-Ca2+ exchanger (NCx), ryanodine receptor (RyR2), and sarcoplasmic reticular Ca2+ ATPase (SERCA2) mRNA levels (expressed in densitometric scores normalized to that of poly(A+) mRNA) in left ventricle from 4-month-old (adult, n = 13) and 24-month-old (senescent, n = 15) rats. For ex vivo contractile function, active tension was measured during isolated heart perfusion in adult (n = 11) and senescent (n = 11) rats. For comparison of age-dependent effects of moderate hypertension on both hemodynamics and calcium proteins, renovascular hypertension was induced or a sham operation performed at 2 (n = 11 and n = 6) and 22 (n = 26 and n = 5) months of age. In senescent rats, LV systolic pressure and maximal rates of pressure development were unaltered, although active tension was depressed (4.7 +/- 0.4 versus 8.3 +/- 0.7 g/g heart weight in adults, P < .0001). SERCA2 mRNA levels were decreased in senescent left ventricle (0.98 +/- 0.05 versus 1.18 +/- 0.05 in adults, P < .01), without changes in NCx and RyR2 mRNA accumulation. Renovascular hypertension resulted in 100% mortality in aged rats; in adults, renovascular hypertension resulted, 2 months later, in an increase of LV systolic pressure (170 +/- 7 versus 145 +/- 3 mm Hg in sham-operated rats, P < .05) and in mild LV hypertrophy (+18%, P < .01) associated with a decrease in SERCA2 mRNA levels (1.02 +/- 0.03 versus 1.18 +/- 0.03 in sham-operated rats, P < .001). Contractile dysfunction in senescent isolated heart and decreased SERCA2 mRNA levels were associated with in vivo normal LV function at rest, indicating the existence of in vivo compensatory mechanisms. RyR2 and NCx gene expressions were not implicated in the observed contractile dysfunction. In aged rats, renovascular hypertension resulted in 100% mortality, probably related to elevated levels of circulating angiotensin II, whereas in adult rats, renovascular hypertension induced a mild LV hypertrophy associated with a selective alteration in SERCA2 gene expression.

Effects of sustained low-flow ischemia on myocardial function and calcium-regulating proteins in adult and senescent rat hearts

OBJECTIVEnBoth aging and myocardial ischemia are associated with alterations of calcium-regulating proteins. We investigated the effects of graded levels of low-flow ischemia on myocardial function and on SR Ca(2+)-ATPase (SERCA2), Na(+)-Ca2+ exchanger (NCX) and ryanodine receptor (RyR2), at mRNA and protein levels in both adult and senescent myocardium.nnnMETHODSnIsolated hearts from 4 and 24 month old (mo) rats were retrogradely perfused during 180 min at 100% (100% CF, n = 11 and n = 11 respectively. 30% (30% CF, n = 10 and n = 12) or 15% (15% CF, n = 13 and n = 8) of their initial coronary flow, and active tension and coronary resistance (in % of their baseline value) were recorded. After 180 min of perfusion. NCX, RyR2 and SERCA2 mRNAs (in % of age-matched 100% CF group value) and protein levels were quantitated in the left ventricles by slot blot and Western blot analysis, respectively.nnnRESULTSnIn 24 mo hearts, low-flow ischemia induced a greater fall in active tension (-65 +/- 7% vs. -40 +/- 4% in 4 mo 30% CF, p, 0.01 and -82 +/- 2% vs. -60 +/- 5% in 4 mo 15% CF groups, p < 0.05 after 15 min of ischemia) and a greater increase in coronary resistance (+357 +/- 44% vs. +196 +/- 39% in 4 mo 30% CF, p < 0.05 and +807 +/- 158% vs. +292 +/- 61% in 4 mo 15% CF groups, p < 0.001 after 15 min of ischemia). An increased accumulation of SERCA2 (+36% and NCX (+46%) transcripts, but not RyR2, already occurred in 24 mo 30% CF group while the 3 transcripts accumulated in 24 mo 15% CF group. In 4 mo rats SERCA2 (+26%), NCX (+35%) and RyR2 (+81%) mRNA levels only increased in the 15% CF group. Corresponding calcium-regulating protein levels were unaltered whatever the degree of flow reduction in both 4 mo and 24 mo hearts.nnnCONCLUSIONnLow-flow ischemia does not induce calcium-regulating protein loss in both adult and senescent hearts. The increase in mRNAs coding for calcium-handling proteins and the impairment of myocardial function which occur at a lesser degree of coronary flow reduction in senescent hearts, indicate a higher vulnerability to low-flow ischemia during aging.

Expression and localization of the annexins II, V, and VI in myocardium from patients with end-stage heart failure.

Annexins II, V, and VI belong to a family of Ca2+-dependent phospholipid-binding proteins that have been involved mainly in signal transduction, differentiation, membrane trafficking events, or binding to the extracellular matrix, or that might be effective as Ca2+-channels. They are abundant in the mammalian myocardium and might play a role in ventricular remodeling and altered calcium handling during heart failure. To test this hypothesis, we compared the expression and distribution of these annexins in nonfailing (n = 9) and failing human hearts with idiopathic dilated cardiomyopathy (n = 11). Northern blot and slot blot analysis were used to determine the annexin mRNA levels and Western blots were used to quantify the amounts of annexin proteins. Distribution of annexins was studied by immunohistofluorescence labeling and compared with that of a sarcolemmal marker (Na+/K+-ATPase) and of a myofibrillar protein (α-actinin). We showed that nonfailing hearts contained a higher amount of annexin VI than of annexin V or II (13.5 ± 1.8, 3.7 ± 0.2, and 2.5 ± 0.5 μg/mg protein, respectively). In failing hearts, there was a parallel increase in both mRNA and protein levels of annexin II (146% and 132%, p < 0.05, respectively) and annexin V (152%, p < 0.01, 147%, p < 0.005, respectively); the protein level of annexin VI was also increased (117%, p < 0.05), whereas the increase of its mRNA level was statistically insignificant. We observed a predominant localization of annexin II in interstitium, and of annexins V and VI in cardiomyocytes at the level of the sarcolemma, T-tubules, and intercalated disks in nonfailing hearts, whereas in failing hearts enlarged interstitium contained all three annexins. Furthermore, annexin V staining at the level of cardiomyocytes almost disappeared. In conclusion, we showed that heart failure is accompanied by marked overexpression of annexins II and V, as well as translocation of annexin V from cardiomyocytes to interstitial tissue. The data suggest that annexins may contribute to ventricular remodeling and annexin V to impaired Ca2+ handling in failing heart.

Inhibition of (Na+ + K+)-ATPase by ouabain: Involvement of calcium and membrane proteins

Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of (Na+ + K+)-stimulated Mg2+-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000-34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca2+. The recovery of the original resistance to ouabain required specifically Ca2+ and was associated with a binding of the solubilized proteins to the membrane.

Modifications of myocardial Na+,K+-ATPase isoforms and Na+/Ca2+ exchanger in aldosterone/salt-induced hypertension in guinea pigs

OBJECTIVEnThe aim of this study was to determine whether changes in cardiac Na+,K(+)-ATPase subunits and Na+/Ca2+ exchanger expression are regulated in aldosterone-salt hypertensive guinea pigs.nnnMETHODSnGuinea pigs (GP) were unilaterally nephrectomized and randomized into three groups (aldosterone-salt; control-salt; control). After 90 days of treatment, echocardiographic M-mode assessment and right carotid arterial catheterization were performed in vivo, and plasma hormones and electrolytes were measured. mRNA and protein levels were studied by Northern and Western blot analysis.nnnRESULTSnAldosterone-salt treatment induced, (1) arterial hypertension (+40%) and LV hypertrophy (+60%) without altering LV-fractional shortening, (2) an increase in plasma norepinephrine levels (+262%) and suppression of renin activity. Northern blot analysis showed the presence of the mRNA encoding the three alpha isoforms and the beta 1 subunit of Na+,K(+)-ATPase in GP myocardium. In the aldosterone-salt group, levels of alpha 1 and beta 1 mRNAs were unchanged. alpha 2 mRNA was increased in both ventricles, whereas alpha 3 mRNA was increased in hypertrophied LV only. Furthermore, levels of the Na+/Ca2+ exchanger mRNA were decreased in both ventricles. At protein level, the two major transcripts (alpha 1 and alpha 2) were detected but alpha 3 isoform was not. Parallel changes in protein and mRNA accumulation of alpha 1 and alpha 2 isoforms were observed in hypertrophied LV.nnnCONCLUSIONnThese results show that alpha 1 and alpha 2 isoforms are expressed in GP heart and that they are independently regulated in aldosterone-salt hypertension. Like the alpha 1 isoform in renal tissue, alpha 2 isoform is the main target of aldosterone-salt. Reciprocal expression of the Na+/Ca2+ exchanger and Na+,K(+)-ATPase suggests an adaptational mechanism which maintains an appropriate sodium gradient and calcium concentration in hypertensive myocardium.

In vivo left ventricular function and collagen expression in aldosterone/salt-induced hypertension

Cardiac fibrosis is linked to aldosterone-induced hypertension, but the effects on in vivo left ventricular (LV) function are not established. We studied the relations between in vivo LV function and aldosterone/salt cardiac fibrosis. Adult guinea pigs (GPs) were treated for 3 months with an aldosterone infusion and high-salt diet. This treatment induced arterial hypertension (+35%) and moderate LV hypertrophy (LVH; +60%) without right ventricular (RV) hypertrophy. Echo-Doppler LV assessment demonstrated unaltered cardiac output, stroke volume, or LV relaxation. Type I collagen messenger RNA (mRNA) was significantly increased in both ventricles (LV, +48%; RV, +77%) and accompanied by a significant increase in total collagen deposition (LV, from 0.52% in controls to 4.4% in treated GPs; RV, from 0.82 to 5.5% in treated GPs). Plasma norepinephrine levels increased 2.6-fold (p < 0.01) and correlated with the increase in collagen deposition in both ventricles. Collagen content was not correlated with hypertension or LVH. We conclude that aldosterone administration induces cardiac collagen accumulation and a sympathetic stimulation, which might preserve systolic and diastolic function.

Involvement of tropomyosin in the sensitivity of Na+ + K+ ATPase to ouabain

Abstract The Na+/K+ ATPase sensitivity to ouabain was shown to be increased by 300 to 1000-fold after treatment of the plasma membrane by EDTA. Addition of proteins detached from the plasma membrane with Ca2+ ions to EDTA treated membranes reconstituted the original Na+/K+ ATPase resistance to ouabain inhibition. Tropomyosin with Ca2+ ions (not with Mg2+ ions) induced the same effect. When suboptimal doses of tropomyosin were used for such a reconstitution, the dose-response curve indicated a full reconstitution of a given percentage of enzyme molecules. This observation led us to assume a direct or indirect effect of tropomyosin on Na+/K+ ATPase functions.

Characterization of a β-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase

Abstract Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of ( Na + + K + )- ATPase to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of M r 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of ( Na + + K + )- ATPase from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the ( Na + + K + )- ATPase . A possible interaction between β-actinin, calmodulin and membrane-bound ( Na + + K + )- ATPase is discussed.

The membrane proteins of the overloaded and senescent heart.

Cardiac hypertrophy which occurs during chronic mechanical overload is one of the numerous examples of biological adaptation to environmental requirements. As such, it is obtained at random by trial and error, and adaptation represents the sum of various modifications in gene expression, including the shift in isoform of myosin or in iso Na+, K+ ATPase, the decrease in beta-adrenergic and muscarinic receptors, ryanodine channels or SR Ca2+ ATPase densities and the unchanged density in Ca2+ current. Some of these changes are beneficial at the cellular level, but are finally detrimental for the organism as a whole, as is the slowing of Vmax. It was suggested that the calcium homeostasis of the hypertrophied cardiocyte was fragile and that this modified cell was less able to buffer the changes in the intracellular calcium, thus providing a biological basis for the arrhythmogenicity of the hypertrophied heart. These various modifications may provide a new key for future pharmaceutical research.

Plasma membrane studies on drug sensitive and resistant cell lines—III: Biphasic kinetics of ouabain binding

Abstract Ouabain binding to wild type and ouabain resistant cell lines derived from the murine plasmocvtoma MOPC 173 was studied on cells grown as monolayers. Biphasic kinetics of ouabain binding was observed; the early phase took place within 30 sec, followed by a spontaneous release of the bound ouabain. Then, the late phase was complete after 1–2 hr. This phenomenon was much more pronounced in fibroblastic than in epithclioid cell lines, regardless of their respective drug sensitivity. Ouabain resistant cells when compared to their wild type counterparts did not exhibit drastic changes either in the number or in the affinity of their respective ouabain binding sites. In contrast, the number and affinity of ouabain binding sites were quite different in fibroblastic and in epithelioid cell lines.