Using the Proteasome to Treat Heart Failure: Protective Effect of PDE1 Inhibition

Using the Proteasome to Treat Heart Failure: Protective Effect of PDE1 Inhibition

Original Paper – PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy: https://advances.sciencemag.org/content/5/5/eaaw5870

Heart disease is the leading cause of death in the United States, and has been for some time. A significant number of these deaths involve heart failure (HF), a clinical condition in which the heart is unable to pump enough blood to keep up with the body’s needs. Heart failure may be categorized into two types according to the heart’s ejection fraction (the percentage of blood out of the total volume of blood in the left ventricle that is pumped out with each contraction): heart failure with reduced ejection fraction (HFrEF), characterized by an ejection fraction of less than 40%, and heart failure with preserved ejection fraction (HFpEF), characterized by normal ejection fraction (50%-70%; 41%-49% considered borderline), with an unhealthy reduction in overall blood volume in the left ventricle. In the United States, about one half of heart failure cases are HFpEF, though the ratio of HFpEF to HFrEF cases has begun to increase, and the former is soon expected to account for the majority of cases. Although significant advancements in treatment for HFrEF have lowered mortality in recent decades, there is still no effective pharmacological treatment for HFpEF, representing a significant unmet medical need.1

            Cardiac proteinopathy is a general category of diseases caused by increased misfolded proteins, and as a result increased proteotoxic stress (IPTS), in heart muscle cells. It is the job of the proteasome to degrade these misfolded proteins to maintain proteostasis and prevent proteotoxic stress. Targeted degradation by the proteasome occurs after the misfolded protein is bound by ubiquitin by a series of co-enzymes. Multiple ubiquitin units are subsequently added, and the 26S proteasome detects and degrades the poly-ubiquitinated protein, preventing them from aggregating. Past studies have implicated proteasome functional insufficiency in cardiomyopathy and progression to heart failure, highlighting proteasomal regulation as a potential target for treatment.2,4

            A previous study by contributor Alfred Goldberg, it was shown that post-translational modification of the 26S proteasome acts as a means of regulation, specifically through the phosphorylation of various subunits, some of which stimulated increased proteasomal activity. Misfolded protein aggregates act as inhibitors to proteasomal activity, making this a promising find in relation to proteostasis maintenance.5 In a later study, Goldberg et al. specifically identified a pathway for proteasome activation by cAMP, via the phosphorylation of the Rpn6 subunit by Protein Kinase A (PKA).6 Author Xuejun Wang identified a similar pathway for proteasomal stimulation by cGMP/PKG.7 These protein kinases are dependent on their respective cyclic nucleotides to function.

            Phosphodiesterase (PDE) is a family of enzymes that break phosphodiester bonds. It is the function of a specific cyclic nucleotide PDE, PDE1, to convert cAMP and cGMP to AMP and GMP respectively, thereby playing an important role in signaling regulation. By breaking down the cyclic nucleotides, PDE1 also effectively inhibits PKA and PKG, and thus their ability to stimulate proteasome activity. Knight and Yan identified PDE1 inhibition as a potential therapy for heart diseases.8 In this current study, the authors demonstrate the effectiveness of PDE1 inhibition in treating HFpEF in mice by upregulating proteasomal activity via increased activation of PKA and PKG.

             In this study, the authors demonstrate that PDE1 inhibition exhibits a protective effect against cardiac proteinopathy and IPTS, and show that this is done via enhanced proteasome activity in a PKG and PKA dependent manner. The experiments utilized a transgenic mouse model of cardiac proteinopathy, induced by the expression of CryABR120G (mutant CryAB), a misfolded mutant form of the protein CryAB known to cause human disease.1

The authors first show that PDE1 protein levels were significantly increased in mutant CryAB mice compared to wild type (healthy) CryAB mice and non-transgenic (NTG; no CryAB) mice. They also demonstrate the ability of PDE1 inhibition to stimulate the ubiquitin-proteasome system, using a green fluorescent protein, GFPdgn. Mice treated with IC86430, a PDE1 inhibitor, showed 40% lower GFPdgn levels than control mice. GFPdgn mRNA levels remained comparable between both groups however, suggesting that the reduced protein levels seen in experimental mice is due to post-transcriptional action.

            Following this, the authors proceed to show the therapeutic effects of PDE1 inhibition on HFpEF. In this experiment, 134 mutant CryAB mice were randomly assigned to two groups, one receiving PDE1 inhibition via IC86430, and the other receiving unaltered vehicle as a control. As expected of mutant CryAB mice, both groups displayed signs of HFpEF prior to treatment. Following the treatment period however, heart malfunction in PDE1-inhibited mice was significantly reversed, with some functions returning to normal expected NTG levels, while the control group did not show the same improvement. Notably, during the 4-week treatment period as well as the 6 weeks following the end of treatment, none of the PDE1-inhibited mice died, while during that same period, 50% of control group mice did die. These results support the ability of PDE1 inhibition to treat cardiac dysfunction, and also potentially implicates the protective effect of PDE1 inhibition as a long term one.

            Experiments were also performed to support the mechanism of the therapeutic effect of PDE1 inhibition, that being the stimulation of proteasome activity by PKG and PKA, following their activation by cGMP and cAMP. After showing that PDE1 inhibition stimulates proteasomal degradation of GFPdgn, a general surrogate substrate of the proteasome, the authors wanted to show that the proteasome would target the disease-causing misfolded mutant CryAB specifically, which they confirmed utilizing a novel protein assay that can distinguish proteasomal degradation of misfolded proteins from other proteins undergoing general turnover. The mutant CryAB mice expectedly showed significantly higher levels of mutant CryAB protein, while NTG mice showed none. PDE1 inhibition significantly lowered mutant CryAB protein levels in mutant mice. Authors also found in this experiment a significant increase in Ser14 phosphorylated Rpn6 subunits in PDE1 inhibited mice, which previous studies have confirmed to occur through PKA. These findings support the proposition that PDE1 inhibition leads to increased levels of PKA, which in turn leads to increased phosphorylation of Rpn6 and thus increased proteasome activity, degrading misfolded proteins in the heart. Finally, the authors demonstrate that this process is dependent on PKA and PKG. Using another green fluorescent protein, GFPu, as the proteasomal substrate, the authors treated mice with a PKG inhibitor (KT5823) and a PKA inhibitor (H89) in addition to the PDE1 inhibitor. Inhibiting PKG and PKA significantly reduced the protective effect of PDE1 inhibition, indicating that this pathway is mediated by PKG and PKA.

            The findings of this paper uncover a new, potentially highly effective and longlasting treatment for heart failure, and in particular, HFpEF, a category of heart failure for which there are currently few therapeutic options. Future research can further explore the ability of PDE1 inhibition to treat cardiac proteinopathy by varying the onset and length of treatment; the beginning of treatment in this study was at the onset of the disease, and the authors predict an even greater effect if treatment is started earlier or longer lasting. Because this study only uses IC86430, similar studies might also be held with other PDE1 inhibitors to determine which might display the most efficacy. This study relates to our course in highlighting the extreme importance of the ubiquitin-proteasome system in preserving normal cell function, as well as the importance of protein quality control in disease prevention.

1. H Zhang, B Pan, P Wu, N Parajuli, MD Rekhter, AL Goldberg, X Wang. PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy. Sci. Adv. 5(5), eaaw5870 (2019). doi: 10.1126/sciadv.aaw5870

2. X Wang, J Robbins. Proteasomal and lysosomal protein degradation and heart disease. J. Mol. Cell. Cardiol. 71, 16–24 (2014). doi: 10.1016/j.yjmcc.2013.11.006

3. E Arbustini, P Morbini, M Grasso, R Fasani, L Verga, O Bellini, B Dal Bello, C Campana, G Piccolo, O Febo, C Opasich, A Gavazzi, VJ Ferrans. Restrictive cardiomyopathy, atrioventricular block, and mild to subclinical myopathy in patients with desmin-immunoreactive material deposits. J. Am. Coll. Cardiol. 31, 645–653 (1998). doi: 10.1016/S0735-1097(98)00026-6

4. J Li, KM Horak, H Su, A Sanbe, J Robbins, X Wang. Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice. J. Clin. Invest. 121, 3689–3700 (2011). doi: 10.1172/JCI45709

5. JJS VerPlank, AL Goldberg. Regulating protein breakdown through proteasome phosphorylation. Biochem. J. 474, 3355–3371 (2017). doi: 10.1042/BCJ20160809

6. JJS VerPlank, S Lokireddy, J Zhao, AL Goldberg. 26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 116, 4228–4237 (2019). doi: https://doi.org/10.1073/pnas.1809254116

7. MJ Ranek, EJM Terpstra, J Li, DA Kass, X Wang. Protein kinase G positively regulates proteasome-mediated degradation of misfolded proteins. Circulation 128, 365–376 (2013). doi: https://doi.org/10.1161/CIRCULATIONAHA.113.001971

8. W Knight, C Yan. Therapeutic potential of PDE modulation in treating heart disease. Future Med. Chem. 5, 1607–1620 (2013). doi:10.4155/fmc.13.127.

10 comments on “Using the Proteasome to Treat Heart Failure: Protective Effect of PDE1 Inhibition

  1. I really like your take on this topic and the paper you used to describe it. It’s very interesting to think about such a common and deadly disease is, to a certain extent, caused by an accumulation of miss-folded proteins. I don’t know if this is something that you took note of in the paper but I’m interested in how the authors induced that miss-folded mutant, how it’s expression was controlled, and what this artificial increase in the concentration of miss-folded protein might have changed in terms of other aspects of the mouse. I guess because they also did a group without this protein at all that chances are the mice were still viable even without this protein so it doesn’t provide the most essential of functions but who really knows.

  2. Hi Ryan, thank you for the interesting paper!! After reading the other posts, it is baffling to me how many novel factors and pathways can be affected by and affect the proteasome (but I suppose they are all connected as all proteins must be degraded at some point). I was really interested in the clinical methods this took in using mice models as well as the larger approach from the standpoint of the relation to heart disease. This is definitely a high impact paper due to its clinical applications. I think it’s also notable that they used mice as well as myocyte trials. I’m curious as to what the actual interaction between PDE1 and IC86430 is, whether it blocks the active site, how it actually outcompetes the substrate and at what rate it is able to hinder the reaction. As it states in the discussion, there could be other metabolites which would more effectively inhibit PDE1, so I’m wondering whether a competitive or allosteric inhibitor would be the next alternative.

    1. Hi Victoria, thanks for the comment! Like I told Brian, I unfortunately couldn’t find much information on the specific interaction between IC86430 and PDE1. The paper didn’t provide any structural or binding information about IC86430, and searching the web doesn’t lead to any databases or other publications that do. One 2006 study by Nagel et al. does provide a table listing the IC50 (inhibitor concentration required for 50% substrate inhibition in vivo) values for various enzymes and their isoforms in the PDE family, but nothing else about the mechanism of inhibition. Future studies I think should definitely provide this information, or determine it graphically if its unknown, as this detail is important in determining which compounds may also have the potential for a similar treatment pathway. Whether or not it is reversible or not, and whether it acts competitively, noncompetitively, or uncompetitively will be important for examining potential side-effects, correct dosage, and efficacy.

  3. Hey Ryan! Great review of this heart disease paper. I have one tissue-level question and one biochemical question. First, why is it that HFpEF is more dangerous than HFrEF? I would expect that HFrEF gets less blood to the body, which would kill brain cells quickly and therefore be more deadly. It seems like there is something else going on in HFpEF patients that is the real cause of heart failure, and if so, what is it?
    Second, what is it about the chemical structure of IC86430 that makes it inhibit PDE1? My gut instinct says it would be a competitive inhibitor, but if it resembles a cNTP then there’s a good chance it’s carcinogenic, so maybe it’s something else entirely. Let me know!

    1. Hey Brian! You’re right about something else going on in HFpEF patients, it also struck me at first that HFrEF would be worse as it seems more apparent that something is malfunctioning. HFpEF seems like it should be pumping more blood as EF is at normal levels. Ejection fraction, reminder, is a measure of the percentage of blood in the left ventricle that gets pumped out with each contraction. Cardiac dysfunction, such as thickening of the heart muscles, leads to the left ventricle having a significantly decreased capacity to hold blood. So even though HFpEF technically has normal EF, the heart failure stems from a lower total volume of blood being pumped. Vic and Zach also asked about the specific mechanism of interaction/inhibition of IC86340 and PDE1 and, unfortunately, I couldn’t seem to find any information about it. I looked through numerous other papers that have used IC86340 as a PDE1 inhibitor and even found the first mention of the compound using google scholar, but none of them seemed to have any such information. For some reason, nobody seems to find it important to mention!

  4. Hey Ryan! Awesome job summarizing a really cool paper with a lot of interesting science going on. Disease involving protein aggregations has been a big focus of mine over the past year in biochemistry, but I have typically only seen this disease occur in the brain, so it’s interesting to see its occurrence elsewhere in the body, as well as the impacts that it has. After reading through, I notice that the authors were very specific in terms of the players which were involved in their experiments. They focused on mutant CryAB, PDE1, as well as IC86430, the PDE1 inhibitor. You pointed out that it would be beneficial to investigate other potential PDE1 inhibitors to see which is most effective. I’m curious, however, about other mutations. Are there other mutations that occur in this variety of heart failure that lead to aggregations? Is CryAB the most common? I’m curious to see if the proteasome is as effective with one set of protein aggregations as opposed to another.

    1. Thanks, Matt! CryAB-R120G (mutant CryAB) was said to be a common cause of heart disease in humans. In this study, the authors used transgenic mutant CryAB mice, meaning the mutant protein was introduced to them artificially. Like most bodily functions, there are probably a number of different causes of proteinopathy-based cardiac dysfunction; the authors merely used one, mutant CryAB, an established human pathogen of the target disease. By demonstrating the effectiveness of PDE1 inhibition-based proteasomal enhancement, the authors imply that most other misfolded or mutant proteins that may play a role in the development of HFpEF will also be targeted by enhanced proteasomal degradation in the same way.

  5. Hi Ryan, this was a great article! I also wrote about the importance of the ubiquitin-proteasome complex for my spotlight blog, and they play such crucial roles that are overlooked by so many people! The research I was involved in last summer was all about heart disease and finding ways to increase angiogenesis, so heart disease research is very important in our current world and it’s so cool to see people using things we learned about in class to solve one of the most prominent diseases in our country. Inhibition of PDE1 was clearly the best route to preventing the aggregation of proteins seen in the 50% death rate in mice without PDE1 inhibition. But for a potential treatment for humans, would there be any severe side-effects from this treatment? Furthermore, what is the mechanism of inhibition of PDE1 by IC86430? This interaction can lead to better understanding of what drugs researchers should develop to help treat this.

  6. Hey Ryan, I really enjoyed reading your post! Heart disease is a really interesting topic, and I think you did a great job with the paper. I have a question about PDE1. The authors describe the PDEs as a family of PDE1-11 with members encoded by distinct genes typically localized to different tissues. With the use of the PDE1 selective inhibitor IC86340, are the authors affecting other isoforms that may be expressed in other tissues? I imagine that there are multiple PDE1 isoforms that may also be inhibited by IC86340, and I am wondering what implications that may have for the rest of the body. Did you come across any information in your background research about any off-target effects of this inhibitor?

    1. Hey Alex, that’s an interesting point you bring up. IC86340 was said to specifically act on the isoforms PDE1A and PDE1C, which are both primarily expressed in the heart, with PDE1A expressed more abundantly in mice and PDE1C more in humans. The authors did not examine tissues other than cardiac muscle. It seems to me that the authors decided this because, with the exception of PDE1B, PDE1 isoforms are not expressed in comparably significant levels in other tissues. Otherwise, this would be, in my opinion, too big an oversight for the authors to draw the specific conclusion that they did. There also did not seem to be any notable clinical manifestations of IC86340 treatment other than the cardio-protective effect observed in this study. The authors in their discussion mention another study by Hashimoto et al, which observed a similar effect in HF dogs using another PDE1 inhibitor, ITI-214, though both inhibitors did have some differences in therapeutic effects. These differences were likely because IC86340 inhibits both PDE1A (which has a higher effinity for cGMP than cAMP) and PDE1C (equal affinity for both), while ITI-214 only inhibits PDE1C. I agree that it certainly would be worth looking at any effects PDE1 inhibition with various inhibitors would have on other parts of the body if any sort of treatment development were to come from this, as there can always be unforeseen effects when altering biochemical pathways.

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