D. Charlemagne

Molecular and cellular biology of the senescent hypertrophied and failing heart

During aging, experimental studies have revealed various cellular changes, principal among which is myocyte hypertrophy, which compensates for the loss of myocytes and is associated with fibrosis. The expression of alpha-myosin heavy chain is replaced by that of the isogene beta-myosin, which leads to decreased myosin adenosine triphosphatase (ATPase) activity. In consequence, contraction is slower and more energetically economical. The Ca(2+)-ATPase of the sarcoplasmic reticulum and Na+/Ca2+ exchange activity are decreased, which probably explains the reduced velocity of relaxation. Membrane receptors are also modified, since the density of both the total beta-adrenergic and muscarinic receptors is decreased. The senescent heart is able to hypertrophy in response to overload and to adapt to the new requirements. Similar alterations are observed both in the senescent heart and in the overloaded heart, in clinical as well as in experimental studies; however, differences do exist, especially in terms of fibrosis and arrhythmias.

The density of ryanodine receptors decreases with pressure overload‐induced rat cardiac hypertrophy

We investigate the possibility that alterations in the calcium movements of the hypertrophied rat heart might involve sarcoplasmic reticulum (SR) ryanodine receptors. A decreased receptor density was observed with severe hypertrophy (0.26±0.05 and 0.35±0.06 pmol/mg protein and 170 and 366 receptors/μm2 of SR in 50–80% hypertrophy and control, respectively); however, the total number of receptors per left ventricle was unchanged. The dissociation constant (0.7 nM) was similar in both hypertrophied and control left ventricles. Thus the decreased density of the ryanodine receptors may participate in altered calcium movements in hypertrophied rat heart.

The effects of compensated cardiac hypertrophy on dihydropyridine and ryanodine receptors in rat, ferret and guinea-pig hearts

The number of dihydropyridine and ryanodine receptors (DHP-R and RyR) has been measured in control and hypertrophied ventricles from rats, guinea pigs and ferrets to determine whether these two channels contribute to the alterations in excitation-contraction coupling (ECC), and in Ca2+ transient during compensated cardiac hypertrophy. We found that ventricular hypertrophy did not change the density of DHP-R. Mild hypertrophy did not alter the density of RyR in the rat but decreased it in the guinea-pig and in the ferret (30% and 36%, respectively). Severe hypertrophy decreased the density of RyR by 20% in the rat and by 34% in the guinea-pig. Therefore, the decrease is greater in ferret and guinea-pig hearts than in rat heart. We conclude that the sarcoplasmic reticulum (SR) Ca2+ release channels but not the L-type Ca2+ channels could contribute to the slowing of intracellular Ca2+ movements and to the reduced velocity of shortening of the hypertrophied hearts. We suggest that, in the guinea pig and ferret hearts which express only the beta myosin heavy chain (MHC) isoform, the reduced velocity of shortening during hypertrophy is related to the decrease in RyR density, whereas in the rat, it is regulated primarily via a shift in the MHC isoform, except in severe hypertrophy in which the moderate decrease in RyR would also be involved.

Expression of laminin α2 chain during normal and pathological growth of myocardium in rat and human

Objectives: Fibrosis is a classical feature of cardiac hypertrophy. To date changes within the basal lamina during normal and pathological cardiac growth have been poorly investigated. The goal of the present study was to determine if the expression of the muscle specific subunit of merosin (laminin α2 chain) together with that of fibronectin (FN) is modified in the diseased human heart. Laminin α2 chain expression was also investigated during physiological and pathological cardiac growth in the rat. Methods: In ten normal human hearts and ten hearts with idiopathic dilated cardiomyopathy (IDCM), the laminin-α2 and FN mRNA levels were quantified by slot-blot using total RNA and the protein distribution was analysed using an immunofluorescence approach. In Wistar rats, laminin α2 and FN mRNA expression was analyzed using RNase protection assay (RPA) and slot-blot assays. Results: The amount of laminin α2 mRNA did not vary in normal and pathological human hearts whereas it was significantly decreased in renovascular hypertensive rats (−20%) P <0.05 versus normal tissue). The amount of fibronectin mRNA increased in IDMC patients (×2, P <0.05 versus normal tissue), but was unchanged in hypertensive rats. A negative correlation was found between the cardiac laminin-α2 level and the age of the patients whatever the cardiac status. During postnatal development in the rat, a similar decrease in cardiac laminin-α2 level was observed between 3 and 30 weeks of age. Finally, the immunofluorescent approach failed to detect any alteration in laminin α2 distribution within the human myocardium. Conclusion: These data indicate that an imbalance between myocyte hypertrophy and the level of laminin-α2 might contribute to alterations in sarcolemmal properties, which occur during the development of cardiac hypertrophy and its transition to cardiac failure.

Cardiac hypertrophy, arrhythmogenicity and the new myocardial phenotype. II. The cellular adaptational process

Ventricular fibrosis is not the only structural determinant of arrhythmias in left ventricular hypertrophy. In an experimental model of compensatory cardiac hypertrophy (CCH) the degree of cardiac hypertrophy is also independently linked to ventricular arrhythmias. Cardiac hypertrophy reflects the level of adaptation, and matches the adaptational modifications of the myocardial phenotype. We suggest that these modifications have detrimental aspects. The increased action potential (AP) and QT duration and the prolonged calcium transient both favour spontaneous calcium oscillations, and both are potentially arrhythmogenic and linked to phenotypic changes in membrane proteins. To date, only two ionic currents have been studied in detail: Ito is depressed (likely the main determinant in AP durations), and If, the pacemaker current, is induced in the overloaded ventricular myocytes. In rat CCH, the two components of the sarcoplasmic reticulum, namely Ca(2+)-ATPase and ryanodine receptors, are down-regulated in parallel. Nevertheless, while the inward calcium current is unchanged, the functionally linked duo composed of the Na+/Ca2+ exchanged and (Na+, K+)-ATPase, is less active. Such an imbalance may explain the prolonged calcium transient. The changes in heart rate variability provide information about the state of the autonomic nervous system and has prognostic value even in CCH. Transgenic studies have demonstrated that the myocardial adrenergic and muscarinic receptor content is also a determining factor. During CCH, several phenotypic membrane changes participate in the slowing of contraction velocity and are thus adaptational. They also have a detrimental counterpart and, together with fibrosis, favour arrhythmias.

Expression of the cardiac ryanodine receptor in the compensated phase of hypertrophy in rat heart

OBJECTIVES Abnormal calcium handling is a general feature of cardiac hypertrophy and alteration in the expression of SR proteins has been suggested to be involved in this alteration. To determine the expression of the cardiac ryanodine receptor (Ry2) gene during compensatory hypertrophy, we studied the mRNA and protein accumulation in left ventricles from rats with 30 to 100% hypertrophy. METHODS Cardiac hypertrophy was obtained after 1 month of aortic constriction. Ry2 mRNA was analyzed by RNase protection assay, Northern and slot blots, and Ry2 protein by high-affinity [3H]ryanodine binding and Western blot. RESULTS We demonstrate that: (1) the cardiac Ry2 mRNA concentration is decreased by 50% in severe hypertrophy; (2) both the density of the high-affinity sites and the Ry2 protein level are decreased by 25%; (3) the decrease in the mRNA and protein levels and the number of high-affinity sites are highly correlated to the severity of hypertrophy. CONCLUSION Our results suggest that, as for SR Ca(2+)-ATPase, there is either a downregulation or a lack of upregulation of the gene coding for the Ry2 in compensatory hypertrophy. The decreased density of Ry2 may alter SR Ca2+ transport and contribute to the impaired Ca2+ handling by slowing the Ca2+ movements.

Localization and quantitation of cardiac annexins II, V, and VI in hypertensive guinea pigs

Annexins are characterized by Ca2+-dependent binding to phospholipids. Annexin II mainly participates in cell-cell adhesion and signal transduction, whereas annexins V and VI also seem to regulate intracellular calcium cycling. Their abundance and localization were determined in left ventricle (LV) and right ventricle (RV) from hypertensive guinea pigs, during the transition from compensatory hypertrophy to heart failure. Immunoblot analysis of annexins II, V, and VI revealed an increased accumulation (2.6-, 1.45-, and 2.3-fold, respectively) in LV from hypertensive guinea pigs and no modification in RV. Immunofluorescent labeling of annexins II, V, and VI; of Na+-K+-ATPase; and of sarcomeric α-actinin showed that in control LV and RV, 1) annexin II is present in nonmuscle cells; 2) annexins V and VI are mainly observed in the sarcolemma and intercalated disks of myocytes; 3) annexins II, V, and VI strongly label endothelial cells and adventitia of coronary arteries; and 4) annexin VI is present in the media. At the onset of heart failure, the most striking changes are the increased protein accumulation in LV and the very strong labeling of annexins II, V, and VI in interstitial tissue, suggesting a role in fibrosis development and cardiac remodeling.

Focal cerebral ischaemia induces a decrease in activity and a shift in ouabain affinity of Na+, K+-ATPase isoforms without modifications in mRNA and protein expression

In a mouse model of focal cerebral ischaemia, we observed after 1 h of ischaemia, that the total Na+, K+-ATPase activity was decreased by 39.4%, and then did not vary significantly up to 6 h post-occlusion. In the sham group, the dose-response curves for ouabain disclosed three inhibitory sites of low (LA), high (HA) and very high (VHA) affinity. In ischaemic animals, we detected the presence of only two inhibitory sites for ouabain. After 1 h of permanent occlusion, the first site exhibited a low affinity while the second site presented an affinity intermediate between those of HA and VHA sites, which evolved after 3 h and 6 h of occlusion towards that of the VHA site. The presence of only two ouabain sites for Na+, K+-ATPase after ischaemia could result from a change in ouabain affinity of both HA and VHA sites (alpha2 and alpha3 isoforms, respectively) to form a unique component. Irrespective of the duration of ischaemia, the smaller activity of this second site accounted entirely for the loss in total activity. Surprisingly, no modifications in protein and mRNA expression of any alpha or beta isoforms of the enzyme were observed, thus suggesting that ischaemia could induce intrinsic modifications of the Na+, K+-ATPase.

Signal and adaptational changes in gene expression during cardiac overload.

Chronic cardiac overload stimulates various quantitative and qualitative mechanisms of adaptation, some of them being species-specific. The signals responsible for these changes in gene expression are still speculative, nevertheless early modifications of the microtubular network have been reported. Soon after overload an increased expression of various genes coding for regulatory proteins has also been observed, this includes various oncogenes and the genes of several heat-shock proteins. Hypertrophy only, is non species-specific and is adaptational because it both multiples the number of contractile units and it lowers wall stress. The slowing of the shortening velocity allows the heart to produce normal tension, at a lower cost, and has different biological explanations depending on the species. In small rodent ventricles, the main but probably not the unique, determinant of this physiological parameter is an isomyosin shift from a high ATPase activity form V1 to a low activity form V3, discovered in our laboratory in 1979. This shift has a transcriptional origin and also occurs in atria in every mammalian including humans; nevertheless it has not been evidenced in the ventricles of humans, dog, cat or guinea-pig. In these species it is necessary to take into account other mechanisms, namely those involved intracellular calcium movements. The number of total, and possibly active, calcium channels is normal in rat overloaded heart suggesting that their synthesis is activated commensurate to the development of hypertrophy. The situation is more complex for other sarcolemma proteins such as the beta-adrenergic system and the Na+, K(+)-ATPase. For the latter there is presently some evidence that an isoenzymatic shift is likely to occur, at least in rats.

Inhibitors of swelling-activated chloride channels increase infarct size and apoptosis in rabbit myocardium.

Apoptosis is a significant contributor to myocardial cell death during ischemia‐reperfusion and swelling‐activated chloride channels (ICl,swell) contribute to apoptosis. However, the relationship between ICl,swell, ischemia‐reperfusion and apoptosis remains unknown. To further investigate this, New Zealand rabbits underwent a 20‐min coronary artery occlusion (CAO) followed by 72 h of coronary artery reperfusion (CAR). Two ICl,swell blockers, 5‐nitro‐2‐[3‐phenylpropylamino]benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA‐94) (both 1 mg/kg), were administered prior to CAO and throughout the 72 h CAR. Infarct size (IS) was increased with NPPB and IAA‐94 compared with control (vehicle) rabbits (51 ± 2% and 48 ± 3% and vs. 35 ± 2%, respectively, P < 0.05). Similar results were found when NPPB was administered only during the reperfusion period. The percentage of TUNEL‐positive nuclei in the border zone of the infarct was increased with NPPB compared with control (37 ± 2% vs. 25 ± 3%, P < 0.05) as well as the number of cytoplasmic histone‐associated DNA fragments (0.45 ± 0.06 vs. 0.33 ± 0.04 absorbance units, P < 0.05). These findings support the concept that ICl,swell channels play an important role in the determination of myocardial infarct size and apoptosis during ischemia‐reperfusion.