Concomitant systolic and diastolic alterations during chronic hypertension in pig.

The mechanical and cellular relationships between systole and diastole during left ventricular (LV) dysfunction remain to be established. LV contraction-relaxation coupling was examined during LV hypertrophy induced by chronic hypertension. Chronically instrumented pigs received angiotensin II infusion for4weeks to induce chronic hypertension (133 ±â€¯7 mmHg vs 98 ±â€¯5 mmHg for mean arterial pressure at Day 28 vs 0, respectively) and LV hypertrophy. LV function was investigated with the instrumentation and echocardiography for LV twist-untwist assessment before and after dobutamine infusion. The cellular mechanisms were investigated by exploring the intracellular Ca2+ handling. At Day 28, pigs exhibited LV hypertrophy with LV diastolic dysfunction (impaired LV isovolumic relaxation, increased LV end-diastolic pressure, decreased and delayed LV untwisting rate) and LV systolic dysfunction (impaired LV isovolumic contraction and twist) although LV ejection fraction was preserved. Isolated cardiomyocytes exhibited altered shortening and lengthening. Interestingly, contraction-relaxation coupling remained preserved both in vivo and in vitro during LV hypertrophy. LV systolic and diastolic dysfunctions were associated to post-translational remodeling and dysfunction of the type 2 cardiac ryanodine receptor/Ca2+ release channel (RyR2), i.e., PKA hyperphosphorylation of RyR2, depletion of calstabin 2 (FKBP12.6), RyR2 leak and hypersensitivity of RyR2 to cytosolic Ca2+ during both contraction and relaxation phases. In conclusion, LV contraction-relaxation coupling remained preserved during chronic hypertension despite LV systolic and diastolic dysfunctions. This implies that LV diastolic dysfunction is accompanied by LV systolic dysfunction. At the cellular level, this is linked to sarcoplasmic reticulum Ca2+ leak through PKA-mediated RyR2 hyperphosphorylation and depletion of its stabilizing partner.

A model of hypoxia-reoxygenation on isolated adult mouse cardiomyocytes: characterization, comparison with ischemia-reperfusion, and application to the cardioprotective effect of regular treadmill exercise.

The use of in vitro experimental models of hypoxia-reoxygenation (H/R) that mimic in vivo ischemia-reperfusion represents a powerful tool to investigate cardioprotective strategies against myocardial infarction. Most in vitro studies are performed using neonatal cardiac cells or immortalized embryonic cardiac cell lines which may limit the extrapolation of the results. We developed an H/R model using adult cardiomyocytes freshly isolated from mice and compared its characteristics to the in vivo ischemia-reperfusion conditions. First, cell death was assessed at different values of pH medium during hypoxia (6.2 vs 7.4) to simulate extracellular pH during in vivo ischemia. Cardiomyocyte mortality was aggravated with hypoxia under acidic pH. We next evaluated the relationship between the duration of hypoxia and cell death. Hypoxia time-dependently reduced myocyte viability (-24%, -36%, -53%, and -74% with 1, 1.5, 2, and 3 hours of hypoxia followed by 17 hours of reoxygenation, respectively). We then focused on the duration of reoxygenation as cardioprotective strategies have been reported to have different effects with short and long durations of reperfusion. We observed that cardiomyocyte mortality was increased when the duration of reoxygenation was increased from 2 h to 17 hours. Finally, we used our characterized model to investigate the cardioprotective effect of regular treadmill exercise. Myocyte viability was significantly greater in exercised when compared to sedentary mice (44% and 26%, respectively). Similarly, mice submitted to in vivo ischemia-reperfusion elicited infarct sizes reaching 27%, 43%, and 55% with 20, 30, and 45 minutes of coronary artery occlusion. In addition, infarct size was significantly reduced by exercise. In conclusion, this H/R model of cardiomyocytes freshly isolated from adult mice shows similar characteristics to the in vivo ischemia-reperfusion conditions. The comparison of in vivo and in vitro settings represents a powerful approach to investigate cardioprotective strategies and to distinguish between direct and indirect cardiomyocyte-dependent mechanisms.

The volume-sensitive chloride channel inhibitors prevent both contractile dysfunction and apoptosis induced by doxorubicin through PI3kinase, Akt and Erk 1/2.

Contractile dysfunction and cardiomyopathies secondary to apoptotic cell death are limiting factors for treating cancer with doxorubicin. Inhibition of volume-sensitive chloride currents (I(Cl,vol)) has been reported to blunt doxorubicin-induced apoptosis in cardiomyocytes. To investigate cellular contractility during acute induction of apoptosis by doxorubicin and to determine whether I(Cl,vol) inhibitors are able to prevent the subsequent contractile dysfunction, electrically paced ventricular myocytes freshly isolated from adult rabbits were acutely exposed to doxorubicin in the presence and absence of I(Cl,vol) inhibitors IAA-94 or DIDS. Doxorubicin induced increases in both annexin V labelling and caspase-3 activity and decreases in cell volume. Alteration in cardiac contractility was observed after doxorubicin exposure. Both IAA-94 and DIDS abolished the doxorubicin-induced decreases in peak shortening and cell volume as well as the increases in caspase-3 activity and annexin V labelling. These protective effects of I(Cl,vol) inhibitors were abolished by previous inhibition of PI(3)kinase, Akt and Erk 1/2. Thus, I(Cl,vol) inhibitors prevent doxorubicin-induced apoptosis and subsequent contractile dysfunction through PI(3)kinase/Akt and Erk 1/2. Inhibition of I(Cl,vol) may represent a new pharmacological strategy for developing cytoprotective drugs against apoptotic cell death and contractile dysfunction.

Volume-sensitive chloride channels (ICl,vol) mediate doxorubicin-induced apoptosis through apoptotic volume decrease in cardiomyocytes.

Apoptosis is associated with early changes in cell volume through a mechanism called apoptotic volume decrease (AVD). As volume-sensitive chloride channels (I(Cl,vol)) are known to play a key role in the regulation of cell volume, this study investigated the role of I(Cl,vol) and AVD in doxorubicin-induced apoptotic cell death in adult rabbit ventricular cardiomyocytes. Exposure of cardiomyocytes to 1 microm doxorubicin induced a rapid and significant reduction in cell volume of cardiomyocytes (average of 15%), i.e. AVD as well as increases in the early markers of apoptosis, annexin V labeling and caspase-3 activity. Doxorubicin also induced the activation of a current characterized as I(Cl,vol) on the basis of the external chloride sensitivity and pharmacological properties with the patch clamp technique. Doxorubicin-induced AVD and apoptosis were both abolished when cardiomyocytes were exposed to the I(Cl,vol) inhibitors 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (0.1 mM) or indanyloxyacetic acid 94 (IAA-94) (10 microM). The crucial role of I(Cl,vol) during AVD and apoptosis was confirmed using C(2)-ceramide, another pro-apoptotic compound. These results demonstrate that activation of I(Cl,vol) plays a major role in the mechanism leading to cell shrinkage and apoptosis-induced AVD by agents such as doxorubicin or C(2)-ceramide in adult cardiomyocytes.

Hypo-osmotic stress inhibits doxorubicin-induced apoptosis via a protein kinase A-dependent mechanism in cardiomyocytes.

1. The clinical use of doxorubicin is limited by the development of severe cardiomyopathies linked, at least in part, to an abnormal increase in the rate of apoptotic cell death. Because cell shrinkage is considered to be a crucial step at the onset of apoptosis, the aim of the present study was to investigate whether a brief hypo-osmotic stress, which leads to an increase in cell volume, could interfere with the induction of apoptosis by doxorubicin in adult cardiomyocytes. 2. Cell volume expansion results in intracellular accumulation of cAMP, so we secondarily tested whether the protective effect of hypo-osmotic stress could be related to the cAMP pathway. Accordingly, apoptosis was induced by doxorubicin (1 micromol/L) in cardiomyocytes freshly isolated from New Zealand adult rabbit hearts. 3. Exposure to doxorubicin in an iso-osmotic medium (290 mOsmol/kg H2O) induced a rapid decrease in cell volume, as well as increases in annexin V labelling and caspase-3 activity, two biological markers of apoptosis. These effects of doxorubicin were abolished by 15 min pretreatment with hypo-osmotic stress at 220 mOsmol/kgH2O (HS 220). 4. This cytoprotective effect of HS 220 was still observed when doxorubicin was added to the medium 60 min later, but it was abolished when the pretreatment by HS 220 was associated with the protein kinase A inhibitor KT 5720 (200 nmol/L). 5. Conversely, 15 min pretreatment with either the cAMP analogue 8-bromo-cAMP (0.5 mmol/L) or the adenylate cyclase activator forskolin (10 micromol/L) inhibited apoptosis induced by doxorubicin. 6. In conclusion, these results demonstrate that: (i) apoptosis induced by doxorubicin can be counteracted by a hypo-osmotic stress in adult cardiomyocytes; and (ii) activation of the protein kinase A-dependent pathway plays a major role in the mechanism leading to the cytoprotective effect induced by a hypo-osmotic stress.

Structure and pharmacology of swelling-sensitive chloride channels, I(Cl,swell).

Since several years, the interest for chloride channels and more particularly for the enigmatic swelling-activated chloride channel (I(Cl,swell)) is increasing. Despite its well-characterized electrophysiological properties, the I(Cl,swell) structure and pharmacology are not totally elucidated. These channels are involved in a variety of cell functions, such as cardiac rhythm, cell proliferation and differentiation, cell volume regulation and cell death through apoptosis. This review will consider different aspects regarding structure, electrophysiological properties, pharmacology, modulation and functions of these swelling-activated chloride channels.

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 (I(Cl,swell)) contribute to apoptosis. However, the relationship between I(Cl,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 I(Cl,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 +/- 31%, 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 I(Cl,swell) channels play an important role in the determination of myocardial infarct size and apoptosis during ischemia-reperfusion.