Research Article: Age-Dependent Protein Expression of Serine/Threonine Phosphatases and Their Inhibitors in the Human Cardiac Atrium

Date Published: January 2, 2019

Publisher: Hindawi

Author(s): Ulrich Gergs, Theresa Trapp, Hasan Bushnaq, Andreas Simm, Rolf-Edgar Silber, Joachim Neumann.


Heart failure and aging of the heart show many similarities regarding hemodynamic and biochemical parameters. There is evidence that heart failure in experimental animals and humans is accompanied and possibly exacerbated by increased activity of protein phosphatase (PP) 1 and/or 2A. Here, we wanted to study the age-dependent protein expression of major members of the protein phosphatase family in human hearts. Right atrial samples were obtained during bypass surgery. Patients (n=60) were suffering from chronic coronary artery disease (CCS 2-3; New York Heart Association (NYHA) stage 1–3). Age ranged from 48 to 84 years (median 69). All patients included in the study were given β-adrenoceptor blockers. Other medications included angiotensin-converting enzyme (ACE) or angiotensin-receptor-1 (AT1) inhibitors, statins, nitrates, and acetylsalicylic acid (ASS). 100 µg of right atrial homogenates was used for western blotting. Antibodies against catalytic subunits (and their major regulatory proteins) of all presently known cardiac serine/threonine phosphatases were used for antigen detection. In detail, we studied the expression of the catalytic subunit of PP1 (PP1c); I1PP1 and I2PP1, proteins that can inhibit the activity of PP1c; the catalytic subunit of PP2A (PP2Ac); regulatory A-subunit of PP2A (PP2AA); regulatory B56α-subunit of PP2A (PP2AB); I1PP2A and I2PP2A, inhibitory subunits of PP2A; catalytic and regulatory subunits of calcineurin: PP2BA and PP2BB; PP2C; PP5; and PP6. All data were obtained within the linear range of the assay. There was a significant decline in PP2Ac and I2PP2A expression in older patients, whereas all other parameters remained unchanged with age. It remains to be elucidated whether the decrease in the protein expression of I2PP2A might elevate cardiac PP2A activity in a detrimental way or is overcome by a reduced protein expression and thus a reduced activity of PP2Ac.

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In the myocardium, Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) via activation of ryanodine receptors is the main mechanism of cardiac excitation-contraction coupling [1]. The ensuing increase in intracellular Ca2+ concentration is responsible for muscle contraction [1]. For relaxation, Ca2+ is mainly removed from the cytosol by the action of SR Ca2+-ATPase (SERCA) into the SR. The affinity of SERCA for Ca2+ is regulated by phospholamban (PLB) located in the SR. Phospholamban itself can be dephosphorylated by two serine/threonine phosphatases, namely, PP1 and PP2A in animal hearts and the human heart [2–5]. The catalytic subunit of PP1 can be inhibited by, amongst others, two endogenous proteins (for review, see [4],, and with peculiar physicochemical properties (preserved action after boiling of samples). These heat stable proteins have been named inhibitor 1 of PP1 (I1PP1 [6]) and inhibitor 2 of PP1 (I2PP1 [6]). Long-term cardiac specific overexpression of I1 leads upon aging in mouse hearts to decreased systolic contractility, suggesting that low PP1 activity is also detrimental for cardiac function in the long run [7]. On the other hand, increased PP1 overexpression in the mouse heart can lead to cardiac hypertrophy and death [8]. In contrast to PP1, another related phosphatase, namely, PP2A, probably mainly exists as a trimer comprising the catalytic subunit, the B subunit, and the A subunit (for current review, see [9]). Cardiac overexpression of PP2A leads in transgenic mice to cardiac hypertrophy [10]. Interestingly, similar to PP1, additional proteins that inhibit PP2A activity have been identified, namely, inhibitor 1 of PP2A (I1PP2A [11]) and inhibitor 2 of PP2A (I2PP2A [12, 13]). Furthermore, a plethora of papers have described and characterized PP2B, consisting of two subunits, termed A subunit and B subunit. The A subunit binds calmodulin and contains the catalytic activity. The B subunit is a Ca2+-binding protein (for review, see [4]). The classical substrate for PP2B in the heart is NFAT which is involved in gene transcription. Much less is known about PP2C in the heart. We have recently generated mice with PP2C overexpression in the heart which led to mild hypertrophy upon aging [14]. It is usually assumed that PP2C is localized in the mitochondria of cardiac cells, and due to this localization it might play a role in ischemia [4]. Finally, PP5 and PP6 are also present in the heart [4], but their functions are not completely understood. At least upon aging, overexpression of PP5 leads to cardiac hypertrophy [15]. There is evidence that the aging human heart exhibits similar contractile defects (due to causative alterations in protein expression) as the failing human heart (end-stage heart failure, NYHA IV [16–19]). For instance, one has noted enhanced activity/expression of PP1 and/or PP2A in end-stage human heart failure [20, 21]. Hence, it is conceivable that protein expression of these major serine/threonine phosphatases might be altered in the aging human heart. Others have studied a role of PP in atrial samples in the development of cardiac failure [22]. Moreover, PP1 activity was increased (but not the protein expression of the catalytic subunit of PP1) in atria in a canine model of tachycardia-induced heart failure [22]; in that model, the Ca content of the SR was enhanced in the atria (but reduced in the ventricle); more Ca could be released from the SR and local basal contractility was enhanced [22], underlining the regional differences in cardiac diseases.

Antibody specificity was first validated using western blotting. Homogenates were prepared from frozen human atrial samples and subjected to western blotting. Linearity of the assay in a range of 25 to 200 µg protein was established initially for all proteins subsequently studied (data not shown) and finally, western blotting experiments were performed using 100 µg proteins per lane. Exemplary full lanes of western blots are also depicted in Figure 1(b), and typical western blots for proteins of interest in typical age groups are presented in Figure 1(a). Similar experiments were done with all other proteins of interest, namely, the catalytic subunit of PP1, the catalytic subunit PP2Ac, the regulatory subunits A and B56α of PP2A, the inhibitory subunits I1PP2A and I2PP2A of PP2A, the inhibitory subunits I1PP1 and I2PP1 of PP1, and the catalytic and regulatory subunits of calcineurin (PP2B), PP5, and PP6. Calsequestrin (CSQ) was used as a loading control as we published before [10, 15]. All antibodies showed specific labeling of proteins with the expected molecular weights (Figure 1). Of note, there was a decreased expression of PP2Ac on protein level in aging (see lanes in the second row from the top in Figure 1(a)) and a decrease of I2PP2A upon aging (see lanes in the second row from bottom in Figure 1(a)). This initial observation was corroborated by studying more samples and performing a statistical analysis (see Figure 2).

Atrial tissue from patients undergoing bypass surgery due to coronary heart disease was studied in the present work. All patients included in this study were on β-adrenoceptor blocker therapy. β-Adrenoceptor blockers can alter many of the biochemical parameters studied here [26]. Several studies on gene expression during aging in animal models [27] and in human tissue also using gene expression arrays [28] have been published. In humans, 162 candidate gene products correlating with heart failure were identified. However, only mRNA for methionine tRNA synthase correlated with age [28]. In nonfailing human hearts, only two transcripts correlated with age, for instance, the abundance of metallothionein 1L increased with age [28]. However, these data were obtained in ventricular tissue and on mRNA levels, whereas we studied atrial tissue and protein expression.




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