Effect of reduced expression of sorcin on Ca2+ handling, in vivo heart function and transcriptional regulation

Malik, Alock Suchitra (2006). Effect of reduced expression of sorcin on Ca2+ handling, in vivo heart function and transcriptional regulation. PhD thesis, Universität zu Köln. Open Access

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Abstract

An adequate and efficient Ca2+ handling is the essential condition for effective functioning of the heart. Decreased peak systolic Ca2+ with prolongation of the duration of Ca2+ transient, slower rates of SR Ca2+ uptake and various other alterations in Ca2+ efflux leading to elevation in diastolic Ca2+ are the key features of the failing heart. These key alterations in heart failure necessitate further evaluation of proteins involved in these regulatory mechanisms. Sorcin, a penta E-F hand family protein associates with cardiac ryanodine receptors, L-type Ca2+ channel as well as SR Ca2+ ATPase and modulates excitation-contraction coupling in the heart. The present thesis aims at understanding the role of sorcin in calcium handling, the effect of decreased level of sorcin on remodeling of the heart and subsequent transcriptional regulation, by using the adenoviral antisense RNA approach. A decrease in the endogenous sorcin expression (75% on the mRNA level and 53% on the protein level) was obtained in the adult rat cardiomyocytes. The decreased amount of sorcin resulted in reduced cell contractility and significantly depressed Ca2+ transient amplitude accompanied with the decreased rate of relaxation in the transfected cardiomyocytes. The increase in stimulation frequency was associated with a negative force-frequency relationship in cardiomyocytes with depressed sorcin, mimicking the behavior exhibited by the failing human cardiomyocytes. However, the β-adrenergic stimulation was unaltered. An oxalate facilitated Ca2+ uptake assay indicated decreased Ca2+ uptake by the sarcoplasmic reticulum in the cardiomyocytes with decreased expression of sorcin. Moreover, incubating the sarcoplasmic reticulum vesicle preparations with recombinant sorcin (1 µM) enhanced the SR Ca2+ uptake and brought it back to the control level. The depressed expression of SR Ca2+ ATPase on the mRNA as well as protein level was also observed. The expression of ryanodine receptors, triadin and phospholamban was unaltered, while a mild increase in the expression of FKBP12.6 was observed. In vivo downregulation of sorcin, achieved by the catheter based cardiac specific gene delivery resulted in severe chamber dilation. Echocardiography revealed ventricular enlargement, decreased heart rate and increased chamber dimension indicating dilated cardiomyopathy in the hearts with depleted levels of sorcin. After 14 days the animals were sacrificed and an increase in the heart weight was observed. In addition, upregulation of calcineurin expression and higher phosphatase activity was observed which was accompanied with increased dephosphorylation of NF-ATC3. GATA4 expression was significantly upregulated in the antisense sorcin transfected cardiomyocytes. The mRNA expression of hypertrophic marker gene β-myosin heavy chain was upregulated, while expression of atrial natriuretic factor and B-type natriuretic peptide was unaltered. In conclusion, using the antisense mRNA approach for sorcin it can be concluded that downregulation of sorcin diminishes the cardiac contractile performance and leads to the ventricular remodeling of the heart. This process is at least partially due to the activation of calcineurin-NFAT signaling pathway. The antisense approach proves to be a valuable tool to identify targets that modulate cardiac contractility and may open new avenues for the treatment of myocardial diseases with diminished cardiac output such as heart failure. An adequate and efficient Ca2+ handling is the essential condition for effective functioning of the heart. Decreased peak systolic Ca2+ with prolongation of the duration of Ca2+ transient, slower rates of SR Ca2+ uptake and various other alterations in Ca2+ efflux leading to elevation in diastolic Ca2+ are the key features of the failing heart. These key alterations in heart failure necessitate further evaluation of proteins involved in these regulatory mechanisms. Sorcin, a penta E-F hand family protein associates with cardiac ryanodine receptors, L-type Ca2+ channel as well as SR Ca2+ ATPase and modulates excitation-contraction coupling in the heart. The present thesis aims at understanding the role of sorcin in calcium handling, the effect of decreased level of sorcin on remodeling of the heart and subsequent transcriptional regulation, by using the adenoviral antisense RNA approach. A decrease in the endogenous sorcin expression (75% on the mRNA level and 53% on the protein level) was obtained in the adult rat cardiomyocytes. The decreased amount of sorcin resulted in reduced cell contractility and significantly depressed Ca2+ transient amplitude accompanied with the decreased rate of relaxation in the transfected cardiomyocytes. The increase in stimulation frequency was associated with a negative force-frequency relationship in cardiomyocytes with depressed sorcin, mimicking the behavior exhibited by the failing human cardiomyocytes. However, the β-adrenergic stimulation was unaltered. An oxalate facilitated Ca2+ uptake assay indicated decreased Ca2+ uptake by the sarcoplasmic reticulum in the cardiomyocytes with decreased expression of sorcin. Moreover, incubating the sarcoplasmic reticulum vesicle preparations with recombinant sorcin (1 µM) enhanced the SR Ca2+ uptake and brought it back to the control level. The depressed expression of SR Ca2+ ATPase on the mRNA as well as protein level was also observed. The expression of ryanodine receptors, triadin and phospholamban was unaltered, while a mild increase in the expression of FKBP12.6 was observed. In vivo downregulation of sorcin, achieved by the catheter based cardiac specific gene delivery resulted in severe chamber dilation. Echocardiography revealed ventricular enlargement, decreased heart rate and increased chamber dimension indicating dilated cardiomyopathy in the hearts with depleted levels of sorcin. After 14 days the animals were sacrificed and an increase in the heart weight was observed. In addition, upregulation of calcineurin expression and higher phosphatase activity was observed which was accompanied with increased dephosphorylation of NF-ATC3. GATA4 expression was significantly upregulated in the antisense sorcin transfected cardiomyocytes. The mRNA expression of hypertrophic marker gene β-myosin heavy chain was upregulated, while expression of atrial natriuretic factor and B-type natriuretic peptide was unaltered. In conclusion, using the antisense mRNA approach for sorcin it can be concluded that downregulation of sorcin diminishes the cardiac contractile performance and leads to the ventricular remodeling of the heart. This process is at least partially due to the activation of calcineurin-NFAT signaling pathway. The antisense approach proves to be a valuable tool to identify targets that modulate cardiac contractility and may open new avenues for the treatment of myocardial diseases with diminished cardiac output such as heart failure.

Item Type:

Thesis (PhD thesis)

Translated title:

Title	Language
Effekt der reduzierten Expression von Sorcin auf Ca2+ handling, in vivo Herzfunktion und transkriptionelle Regulation	German

Translated abstract:

Abstract

Language

Für eine effektive Herzfunktion ist eine effiziente Ca2+-Homöostase wesentlich. Vermindert systolisch freigesetztes Ca2+, eine Verlängerung des Ca2+-Transienten und eine reduzierte Aufnahmerate des sarkoplasmatischen Retikulums (SR) sind wesentliche Veränderungen einer Herzinsuffizienz. Diese Störungen machen eine weitere Untersuchung von Proteinen notwendig, die die myokardiale Ca2+-Homöostase regulieren. Sorcin ist ein EF-Hand Protein, dass mit dem kardialen Ryanodin-Rezeptor, dem L-Typ Ca2+-Kanal sowie mit der SR Ca2+-ATPase assoziiert ist und die elektromechanische Kopplung am Herzen mitreguliert. Die vorliegende Doktorarbeit hatte zum Ziel, die Rolle von Sorcin für die Ca2+-Homöostase, die Bedeutung einer verminderten Expression von Sorcin auf die Herzentwicklung und die transkriptionale Regulation mit Hilfe von adenoviralem Antisense Sorcin zu untersuchen. Eine Reduktion der endogenen Sorcin Expression (74% der mRNA Expression und 53% der Proteinexpression) konnte mit Hilfe dieses adenoviralen antisense Ansatzes in isolierten Rattenkardiomyozten erzielt werden. Die verminderte Sorcin Expression führte zu einer reduzierten Zellkontraktion, einer verminderten Amplitude des Ca2+-Transienten und einer Verlängerung der Relaxationsrate. Erhöhung der Stimulationsfrequenz zeigte eine negative Kraft-Frequenz-Beziehung (KFB) in Kardiomyozyten mit verminderter Sorcin Expression, die mit der KFB an insuffizienten, menschlichen Kardiomyozyten vergleichbar ist. Die b-adrenerge Stimulation blieb erhalten. Der Ca2+-Uptake mit Oxalat zeigte verminderte Aufnahmeraten des SR an Kardiomyozyten mit verminderter Sorcin Expression, Inkubation mit rekombinantem Sorcin (1µM) führten zu einer erhöhten SR Ca2+-Aufnahmerate. Zudem wurde eine reduzierte Expression der SR Ca2+-ATPase nachgewiesen. Die Expression des Ryanodin Rezeptors, von Triadin und Phospholamban blieb unverändert, während die Proteinexpressin von FKBP 12,6 erhöht war. Reduktion der Expression von Sorcin mit Hilfe eines katheterbasierten, intrakoronaren Applikationsweges in vivo führte zu einer schweren Herzdilatation. Echokardiographie der transfizierten Herzen zeigte eine ventrikuläre Herzvergrösserung, eine verminderte Herzfrequenz und einen erhöhten end-diastolische Durchmesser. Nach 14 Tagen Transfektion wurde morphologisch eine Zunahme des Herzgewichtes gesehen. Zudem wurden an Antisense Sorcin transfizierten Kardiomyozyten eine erhöhte Calcineurin Aktivität und Expression, verbunden mit einer höheren NF-ATC3 Dephosphorylierung, und eine erhöhte Expression der GATA4 Expression gefunden. Das Hypertrophiemarker-Gene "β- Myosin Heavy Chain" war erhöht, während die Expression des atrialen, natiuretischen (ANP) und gehirnspezifischen, natiuretischen Peptiden (BNP) unverändert blieb. Zusammenfassend werden durch eine Verminderung der Sorcin Expression mit Hilfe eines adenoviralen Anti-sense Ansatzes eine reduzierte kardiale Kontraktionskopplung und eine dilatative Kardiomypathie ausgelöst. Dieser Prozess beinhaltet Störungen der Ca2+-Homöostase und Aktivierung des Calcineurin- NFAT Signaltransduktionsweges. Die adenovirale anti-sense Strategie kann dazu verwandt werden, um Zielproteine bei der Entstehung einer Herzinsuffizienz zu identifizieren, die die kardialen Kontraktilität und Leistung insbesondere bei verminderten kardialen Pumpleistung verbessern können. Dieser Ansatz kann in der Entwicklung neuer Therapieformen bei der menschlichen Herzinsuffizienz verwandt werden.

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