Alterations in calcium (Ca) homeostasis play a major role in the pathophysiology of myocardial ischemia and of heart failure. Since the ACE-inhibitor zofenopril is known to be beneficial both in heart failure and in acute ischemia, we investigated whether it may modulate the expression of genes involved in the regulation of calcium homeostasis. We used an acute and a chronic model. In the acute model isolated rat hearts were perfused for 120 minutes in the presence or in the absence of 10 microM zofenoprilat, the active metabolite of zofenopril. At the end of perfusion, each heart was homogenized, RNA was extracted and RT-PCR was used to evaluate the expression of the following genes: sarcolemmal L-type Ca channel (CaV), sarcolemmal Na-Ca exchanger (NCE), sarcoplasmic reticulum Ca channel/ryanodine receptor (RyR), sarcoplasmic reticulum Ca-ATPase (SERCA), SERCA modulator phospholamban (PLB). Gene expression was normalized on the basis of glyceraldehyde-3-phosphate dehydrogenase expression. In parallel experiments the heart was homogenized at the end of perfusion and oxalate-supported 45Ca uptake, which provides a reliable estimate of sarcoplasmic reticulum calcium uptake, was determined. In chronic experiments rats were treated with zofenopril (15 mg/kg die per os) for 14 days, while control hearts were treated with the same diet, except that zofenopril was omitted. At the end of this period, the heart was quickly excised and perfused with the working heart technique, then it was homogenized and oxalate-supported 45Ca uptake was determined. Acute or chronic zofenopril administration did not produce any significant change in cardiac output, developed pressure, heart rate, or coronary flow. RT-PCR experiments showed that after 120 min of exposure to 10 microM zofenoprilat PLB gene expression was significantly decreased (0.60±0.04 vs 0.78±0.05 arbitrary units, P<0.05), while the expression of CaV, NCX, RyR and SERCA genes was not significantly modified. Functional experiments revealed that the rate of oxalate-supported 45Ca uptake measured at 1 micromolar free calcium concentration was significantly increased both in the acute (11.7±0.5 vs 14.1±0.4 nmol/min per mg of protein, P<0.01) and in the chronic model (10.4±0.8 vs 14.5±0.7 nmol/min per mg of protein, P<0.01). We conclude that acute or chronic exposure to zofenopril increased sarcoplasmic reticulum calcium uptake, possibly as a consequence of reduced PLB expression. This action might contribute to the beneficial effects of zofenopril in heart failure and in ischemic heart disease.

Effect of acute and chronic zofenopril administration on cardiac calcium homeostasis

GHELARDONI, SANDRA;CHIELLINI, GRAZIA;ZUCCHI, RICCARDO
2009

Abstract

Alterations in calcium (Ca) homeostasis play a major role in the pathophysiology of myocardial ischemia and of heart failure. Since the ACE-inhibitor zofenopril is known to be beneficial both in heart failure and in acute ischemia, we investigated whether it may modulate the expression of genes involved in the regulation of calcium homeostasis. We used an acute and a chronic model. In the acute model isolated rat hearts were perfused for 120 minutes in the presence or in the absence of 10 microM zofenoprilat, the active metabolite of zofenopril. At the end of perfusion, each heart was homogenized, RNA was extracted and RT-PCR was used to evaluate the expression of the following genes: sarcolemmal L-type Ca channel (CaV), sarcolemmal Na-Ca exchanger (NCE), sarcoplasmic reticulum Ca channel/ryanodine receptor (RyR), sarcoplasmic reticulum Ca-ATPase (SERCA), SERCA modulator phospholamban (PLB). Gene expression was normalized on the basis of glyceraldehyde-3-phosphate dehydrogenase expression. In parallel experiments the heart was homogenized at the end of perfusion and oxalate-supported 45Ca uptake, which provides a reliable estimate of sarcoplasmic reticulum calcium uptake, was determined. In chronic experiments rats were treated with zofenopril (15 mg/kg die per os) for 14 days, while control hearts were treated with the same diet, except that zofenopril was omitted. At the end of this period, the heart was quickly excised and perfused with the working heart technique, then it was homogenized and oxalate-supported 45Ca uptake was determined. Acute or chronic zofenopril administration did not produce any significant change in cardiac output, developed pressure, heart rate, or coronary flow. RT-PCR experiments showed that after 120 min of exposure to 10 microM zofenoprilat PLB gene expression was significantly decreased (0.60±0.04 vs 0.78±0.05 arbitrary units, P<0.05), while the expression of CaV, NCX, RyR and SERCA genes was not significantly modified. Functional experiments revealed that the rate of oxalate-supported 45Ca uptake measured at 1 micromolar free calcium concentration was significantly increased both in the acute (11.7±0.5 vs 14.1±0.4 nmol/min per mg of protein, P<0.01) and in the chronic model (10.4±0.8 vs 14.5±0.7 nmol/min per mg of protein, P<0.01). We conclude that acute or chronic exposure to zofenopril increased sarcoplasmic reticulum calcium uptake, possibly as a consequence of reduced PLB expression. This action might contribute to the beneficial effects of zofenopril in heart failure and in ischemic heart disease.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/129532
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