Regulation of cardiomyocyte DNA damage and cell death by the type 2A protein phosphatase regulatory protein alpha4

The type 2A protein phosphatase regulatory protein alpha4 (α4) constitutes an anti-apoptotic protein in non-cardiac tissue, however it’s anti-apoptotic properties in the heart are poorly defined. To this end, we knocked down α4 protein expression (α4 KD) using siRNA in cultured H9c2 cardiomyocytes and confirmed the lack of DNA damage/cell death by TUNEL staining and MTT assay. However, α4 KD did increase the phosphorylation of p53 and ATM/ATR substrates, decreased the expression of poly ADP-ribose polymerase and associated fragments. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xL was reduced, whereas expression of pro-apoptotic BAX protein did not change. Alpha4 KD reduced basal H2AX Ser139 phosphorylation, whereas adenoviral-mediated re-expression of α4 protein following α4 KD, restored basal H2AX phosphorylation at Ser139. The sensitivity of H9c2 cardiomyocytes to doxorubicin-induced DNA damage and cytotoxicity was augmented by α4 KD. Adenoviral-mediated overexpression of α4 protein in ARVM increased PP2AC expression and augmented H2AX Ser139 phosphorylation in response to doxorubicin. Furthermore, pressure overload-induced heart failure was associated with reduced α4 protein expression, increased ATM/ATR protein kinase activity, increased H2AX expression and Ser139 phosphorylation. Hence, this study describes the significance of altered α4 protein expression in the regulation of DNA damage, cardiomyocyte cell death and heart failure

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium

Enzyme inhibition as a potential therapeutic strategy to treat COVID-19 infection

With the emergence of the third infectious and virulent coronavirus within the past two decades, it has become increasingly important to understand how the virus causes infection. This will inform therapeutic strategies that target vulnerabilities in the vital processes through which the virus enters cells. This review identifies enzymes responsible for SARS-CoV-2 viral entry into cells (ACE2, Furin, TMPRSS2) and discuss compounds proposed to inhibit viral entry with the end goal of treating COVID-19 infection. We argue that TMPRSS2 inhibitors show the most promise in potentially treating COVID-19, in addition to being a pre-existing medication with fewer predicted side-effects

Saponification of carboxylmethylated PP2A_C by alkalinisation.

<p>ARVM were exposed to the A1.R agonist CPA (0–100 µM) for 10 minutes and PP2A_C carboxylmethylation was detected by Western immunoblotting (IB) with either an anti-methyl PP2A_C (2A10) or demethylated PP2A_C (4B7) antibody before (−NaOH) or after (+NaOH) saponification of the C-terminal leu309 methylation by 100 mM NaOH. (B) Carboxylmethylation of PP2A_C in response to CGP (10 nM), CPA (10 µM) or CCH (10 µM) as detected by Western immunoblotting with either an anti-methyl PP2A_C (2A10) or demethylated PP2A_C (4B7) antibody with or without treatment with 100 mM NaOH. Immunoblots are representative of 3 individual experiments.</p

Subcellular localisation of proteins that regulate PP2A_C carboxylmethylation and targeting in ARVM.

<p>ARVM were initially lysed with a digitonin-based buffer to separate the cytoplasm and particulate fractions. The particulate fraction was then resuspended with a Triton X-100 based buffer to separate the soluble membrane and insoluble fractions by centrifugation. Samples were then analysed by standard Western immunoblotting (IB). (A) Subcellular localisation of PP2A_C and B55α protein in the cytoplasm (CYTO), soluble membrane (SOL) and insoluble (INSOL) fractions in ARVM. The presence of cardiac troponin I (cTnI) protein was to confirm the purity of the insoluble (INSOL) fraction. Subcellular localisation of PME-1 (B) and LCMT-1 (C) protein in the whole cell lysate (WCL), cytoplasm (CYTO), soluble membrane (SOL) and insoluble (INSOL) fractions in ARVM. All columns represent mean optical density (OD) values ± SEM, n = 3 individual experiments.</p

Role of Gβγ subunits in CPA-induced PP2A_C translocation.

<p>ARVM were lysed with a digitonin-based buffer to separate the cytoplasm and particulate fractions by centrifugation. Samples were then saponified with NaOH to abrogate any PP2A_C carboxylmethylation. (A) Total PP2A_C content in the particulate fraction of ARVM in response to increasing concentrations of CPA (0–100 µM) was indexed by Western immunoblotting (IB) with an anti-demethylated PP2A_C antibody (4B7) following treatment with 100 mM NaOH. Total PP2A_C content in the particulate fraction was quantified by densitometry. (B) Multiplicity of infection (MOI)-dependent co-expression of EGFP and Gα_t1 protein in ARVM infected with the AdV:Gα_t1. (C) ARVM were infected with either the control AdV:EGFP or AdV:Gα_t1 for 18 hours and then exposed to10 µM CPA for 10 minutes. ARVM were then lysed and total PP2A_C content in the particulate fraction was indexed by Western immunoblotting (IB) with an anti-demethylated PP2A_C (4B7) antibody following treatment of samples with 100 mM NaOH. PP2A_C content in the particulate fraction was quantified by densitometry. All columns represent mean values ± SEM, n = 4 individual experiments, *p<0.05 vs 0 (control group).</p

G_iPCR-induced carboxylmethylation of PP2A_C in ARVM.

<p>ARVM were exposed to the G_iPCR agonists (A) CPA (0–100 µM), (B) CGP (0–100 nM) or (C) CCH (0–100 µM) for 10 minutes. Carboxylmethylation of PP2A_C (as % of vehicle control) was detected by Western immunoblotting (IB) of the whole cell lysate with either an anti-methyl PP2A_C (2A10) or an anti-PP2A_C FL-309 (FL) antibody to determine equal protein loading. PP2A_C carboxylmethylation was quantified by densitometry and all columns represent mean values ± SEM, n = 4–6 individual experiments, *p<0.05 vs 0 (control group).</p

Suggested intracellular signalling mechanism (s) through which A1.Rs induce PP2A_C translocation.

<p>Our data suggests that the stimulation of G_i protein-coupled adenosine A1 receptors by the agonist CPA increases the association of PP2A_C with LCMT-1, thereby augmenting the leucine carboxylmethylation status of PP2A_C. The stimulation of G_i protein-coupled adenosine A1 receptors by the agonist CPA also elicits a cascade involving the release of Gβγ subunits which activate PI3K. Both of these intracellular signalling events coordinate and facilitate the association of PP2A_C with the membrane-rich particulate compartment of ARVM.</p