Efficacy and Safety of Angiotensin Receptor Blockers in a Pre-Clinical Model of Arrhythmogenic Cardiomyopathy

Arrhythmogenic Cardiomyopathy (ACM) is a familial heart disease, characterized by contractile dysfunction, ventricular arrhythmias (VAs), and the risk of sudden cardiac death. Currently, implantable cardioverter defibrillators and antiarrhythmics are the mainstays in ACM therapeutics. Angiotensin receptor blockers (ARBs) have been highlighted in the treatment of heart diseases, including ACM. Yet, recent research has additionally implicated ARBs in the genesis of VAs and myocardial lipolysis via the peroxisome proliferator-activated receptor gamma (PPARγ) pathway. The latter is of particular interest, as fibrofatty infiltration is a pathological hallmark in ACM. Here, we tested two ARBs, Valsartan and Telmisartan, and the PPAR agonist, Rosiglitazone, in an animal model of ACM, homozygous Desmoglein-2 mutant mice (Dsg2mut/mut). Cardiac function, premature ventricular contractions (PVCs), fibrofatty scars, PPARα/γ protein levels, and PPAR-mediated mRNA transcripts were assessed. Of note, not a single mouse treated with Rosiglitazone made it to the study endpoint (i.e., 100% mortality: n = 5/5). Telmisartan-treated Dsg2mut/mut mice displayed the preservation of contractile function (percent ejection fraction [%EF]; 74.8 ± 6.8%EF) compared to Vehicle- (42.5 ± 5.6%EF) and Valsartan-treated (63.1 ± 4.4%EF) mice. However, Telmisartan-treated Dsg2mut/mut mice showed increased cardiac wall motion abnormalities, augmented %PVCs, electrocardiographic repolarization/depolarization abnormalities, larger fibrotic lesions, and increased expression of PPARy-regulated gene transcripts compared to their Dsg2mut/mut counterparts. Alternatively, Valsartan-treated Dsg2mut/mut mice harbored fewer myocardial scars, reduced %PVC, and increased Wnt-mediated transcripts. Considering our findings, caution should be taken by physicians when prescribing medications that may increase PPARy signaling in patients with ACM.


Introduction
Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease associated with progressive muscle deterioration, lethal arrhythmias, exercise-induced disease penetrance, inflammation, and fibrofatty replacement of the myocardium [1]. Known as a "disease of the cardiac desmosome," the majority of clinical reports are linked to inherited pathogenic variants (primarily, autosomal dominant) in desmosomal genes, such as PKP2 (Plakophilin-2) and DSG2 (Desmoglein-2) [1]. Despite experimental and clinical evidence on the efficacy of current pharmacological treatment options, such as antiarrhythmics and angiotensin receptor blockers (ARBs), little is known regarding ARBs' role in the origin of ventricular arrhythmias and increased myocardial lipolysis via the peroxisome proliferator-activated receptor (PPAR) pathway. Kim, C. et. al. demonstrated in induced pluripotent stem-cellderived cardiomyocytes (iPSC-CMs) from a patient harboring a pathogenic PKP2 variant that introducing exogenous PPARγ caused a shift in metabolic respiration, increased lipolysis and apoptosis, decreased β-catenin activity, and abnormal calcium handling [2]. In the 2 of 11 present study, we used an animal model of ACM-homozygous Desmoglein-2 mutant mice (Dsg2 mut/mut ) [3][4][5]. This is a robust animal model of ACM that recapitulates the disease time course seen in patients with ACM. Specifically, Dsg2 mut/mut mice are asymptomatic at birth and show subclinical phenotypes (e.g., ECG depolarization/repolarization abnormalities) until 8 weeks of age [3][4][5] whereby, at 8 weeks of age, echocardiographic analyses demonstrate mild dysfunction [3][4][5]. From 8 to 16 weeks of age, large-scale myocyte cell death and both innate and extrinsic inflammation lead to pathological progression [3][4][5], whereby, at 16 weeks of age, Dsg2 mut/mut mice demonstrate numerous PVCs, severe cardiac dysfunction, and biventricular fibrosis (Supplementary Figure S1) [3][4][5].
Thus, Dsg2 mut/mut mice were utilized in this study to provide further insight into the efficacy and safety of two specific ARBs with discordant effects on PPAR signaling, Valsartan and Telmisartan, as a pharmacological therapeutic in ACM. Moreover, we treated mice with Rosiglitazone, a selective PPARγ agonist with no PPARα-binding affinity to test whether drugs with PPARγ agonism (i.e., Telmisartan or Rosiglitazone) worsen ACM phenotypes.
Recent studies demonstrated the deleterious effects of PPARγ signaling in the pathophysiology of ACM progression [2,6]. However, these prior studies lack in vivo testing of thiazolidinediones (i.e., PPARγ agonists) in animal models of ACM. Our study reveals the detrimental impact of PPARγ agonism in ACM mice, as a 100% mortality rate occurred in Rosiglitazone-treated Dsg2 mut/mut mice before the study endpoint. Yet, even at the study midpoint, Rosiglitazone-treated Dsg2 mut/mut mice had significant cardiac dysfunction. Caution is warranted when prescribing ARBs to ACM patients, such as those with comorbidities (e.g., diabetes mellitus type-2). Even so, thiazolidinediones have been known to cause fluid retention and thus may complicate treatment with ACM patients developing or with developed heart failure. Alternatively, while Valsartan-treated Dsg2 mut/mut mice did not delay the progression of cardiac dysfunction, it normalized QRS duration, reduced ventricular ectopy, and prevented further fibrotic progression. Interestingly, Valsartantreated Dsg2 mut/mut mice showed increased nuclear PPARα levels and increased active β-catenin with elevated Wnt/β-catenin transcripts, the latter of which is down-regulated in ACM [7].
In summary, this article provides evidence that Valsartan is a safe and effective ARB to reduce ventricular ectopy and ECG repolarization and depolarization abnormalities, prevent fibrofatty scar formation, and increase Wnt-mediated gene transcription. More studies should be conducted regarding drug titration levels of Valsartan to acquire the optimal dose for the preservation of cardiac function without adverse outcomes. Although Telmisartantreated Dsg2 mut/mut mice did show the preservation of cardiac function, caution should be implemented for providers in prescribing Telmisartan, especially considering our results indicate worsening of arrhythmic burden, a variety of cardiac wall motion abnormalities, and increased fibrotic remodeling in Telmisartan-treated Dsg2 mut/mut mice.
The damaging effects of PPARγ agonism in ACM have been previously demonstrated [2,6], yet to our knowledge, this has yet to be tested in vivo. Therefore, we treated Dsg2 mut/mut mice with Rosiglitazone (Ros), a thiazolidinedione that specifically acts as a PPARγ agonist. Of monumental importance, Ros-treated Dsg2 mut/mut mice exhibited a 100% mortality rate by 9 weeks of age ( Figure 1A). Considering the rapid mortality rate in Ros-treated Dsg2 mut/mut mice, we collected Echos from the remaining n = 3 mice at 7W in the event that more deaths occurred in this cohort. We observed a stark decrease in cardiac function and aberrant cardiac remodeling in Ros-treated Dsg2 mut/mut mice vs. Veh-treated Dsg2 mut/mut , Veh-treated WT, and Ros-treated WT cohorts at 7 weeks of age (Supplementary Figure S2A-D mouse died 3 weeks prior (overall cohort survival = 85.7%; Figure 1A) to the Veh-treated Dsg2 mut/mut mouse that died. No deaths occurred in Veh-treated WT mice ( Figure 1A).  . Data presented as mean ± SEM. * p < 0.05 for any cohort vs. Vehicle-treated WT mice; † p < 0.05 for any drug-treated Dsg2 mut/mut cohort vs. Vehicle-treated Dsg2 mut/mut mice; & p < 0.05 for Valsartan-treated Dsg2 mut/mut mice vs. Telmisartan-treated Dsg2 mut/mut mice, using One-way ANOVA with Tukey's post-hoc analysis. # p < 0.05 any cohort vs. Rosiglitazone-treated Dsg2 mut/mut mice using Mantel-Cox and Wilcoxon survival analysis. Blue, WT vehicle-treated mice; pink, Dsg2 mut/mut Rosiglitazone-treated mice; black, Dsg2 mut/mut Valsartan-treated mice; green, Dsg2 mut/mut Telmisartan-treated mice; and red, Dsg2 mut/mut vehicle-treated mice.
At the study midpoint (8W) and endpoint (16W) (Supplementary Table S1 and Table 1, respectively), echocardiographic, ECG, and blood pressure plethysmography (BPP) data were collected. Dsg2 mut/mut mice treated with either Veh or Val presented with reduced cardiac function (%EF) at 16W compared to Veh-treated WT mice ( Figure 1B; Table 1). That said, Val-treated Dsg2 mut/mut mice still showed improved cardiac function compared to Veh-treated Dsg2 mut/mut mice ( Figure 1B, Table 1). Conversely, Tel-treated Dsg2 mut/mut mice showed the preservation of cardiac function compared to Veh-and Val-treated Dsg2 mut/mut counterparts ( Figure 1B; Table 1). Despite this finding, Echo analyses in Tel-treated Dsg2 mut/mut mice demonstrated that this cohort presented with hypokinesia, dyskinesia, and aneurysms of the left ventricular anterior and posterior free walls ( Figure 1C, D; Table 1). Next, signal-average ECGs (SAECGs) showed that Val-and Teltreated Dsg2 mut/mut mice displayed shortening of the RR-Interval (RR-I) and increased heart rate (HR) compared to Veh-Dsg2 mut/mut mice ( Figure 1E,F; Table 1). Although Veh-Dsg2 mut/mut mice showed a trend towards prolonged QRS-duration (QRSd), only Tel-treated Dsg2 mut/mut mice showed increased QRSd compared to WT mice ( Figure 1F; Table 1). Additionally, Tel-treated Dsg2 mut/mut mice demonstrated augmented premature ventricular contractions (PVCs; Figure 1G) and an increased Q-amplitude ( Figure 1E; Table 1); thus, Telmisartan appears to cause ventricular depolarization abnormalities in ACM mice. As a vasodilator, Telmisartan exhibited a nearly 30% reduction in systolic, diastolic, and mean blood pressure compared to Veh-and Val-treated Dsg2 mut/mut mice (Table 1). A similar reduction in blood pressure was observed in Tel-treated WT mice, yet Valsartan appeared to have no effect in WT mice.

Parameters
Lastly, ARB-induced gene transcription was analyzed for changes in PPARs, PPAR coregulators, and metabolism and lipid transcripts ( Figure 2H and Supplementary Figure S2E,F).
The most interesting results uncovered were that both Ppar α , Ppar δ , and PPARγ co-activator-1α (Pgc1α) and -1β (Pgc1β) expression was elevated in Tel-treated Dsg2 mut/mut mice ( Figure 2H). This was particularly interesting considering PPARγ nuclear protein levels were reduced in these same mice. Of note, pyruvate dehydrogenase (Pdh) and Pdh kinase isoform-2 (Pdk2) expressions were up-regulated in Val-treated Dsg2 mut/mut mice compared to Veh-treated Dsg2 mut/mut mice (Supplementary Figure S2E). Intriguingly, the top transcription factor binding sites for Pdh and Pdk2 gene promoters are c-Myc and Nfat1-4, whereby the former is a primordial Wnt/β-catenin transcript and the latter increases the transcription of the Frizzled-A Wnt co-receptor [8]. These findings are certainly intriguing given that Val-treated Dsg2 mut/mut mice showed elevated levels of β-catenin (Ctnnb1; Figure 2H). There was a stark decline in Pdk4 expression, an endogenous inhibitor of Pdh, in Val-treated Dsg2 mut/mut mice compared to Vehtreated Dsg2 mut/mut mice (Supplementary Figure S2E). Although the expression of adiponectin (Adipo) was elevated in nearly all Dsg2-cohorts (Supplementary Figure S2F), only Val-treated Dsg2 mut/mut mice showed reduced levels of the adipokine, fatty acid binding protein-4 (Fabp4; Supplementary Figure S2F). In conclusion, it appears Valsartan increases gene transcripts to catabolize fat into energy (i.e., ATP) through the TCA cycle, in addition to increasing Wnt/β-catenin transcription.

Discussion
Considering that fibrofatty replacement of the myocardium is a pathological hallmark of ACM, we were interested in the effects of thiazolidinediones (i.e., Rosiglitazone) and/or partial-thiazolidinediones (i.e., Telmisartan), on cardiac function, lipolytic gene expression, and fibrofatty infiltration. The ARB, Telmisartan, was of particular interest given recent work that demonstrated it harbors partial-PPARγ agonism [9]. This is crucial in ACM given that the suppression of the Wnt/β-catenin signaling pathway is implicated in ACM pathogenesis [7] and PPARγ and Wnt share an inverse relationship with one another. Specifically, PPARs work in complete opposition to Wnt, such that if PPARs are up-regulated then Wnt is down-regulated, and vice versa [10].
As an ARB, Telmisartan exceeded the blood pressure reduction compared to Valsartan at both 8 and 16 weeks of age. Additionally, Telmisartan preserved left ventricular function to nearly WT levels. However, Telmisartan-treated Dsg2 mut/mut mice showed increased wall motion abnormalities, myocardial fibrosis, PVCs, and depolarization abnormalitiesall Task Force Criteria phenotypes for ACM. Alternatively, while Valsartan preserved cardiac function, it was nearly devoid of conduction abnormalities, arrhythmias, and reduced RVF, and myocardial samples showed increased PPARα nuclear levels, active β-catenin, and Wnt-mediated mRNA transcription. Prior work demonstrated PPARα activation is necessary to combat ROS-mediated cellular damage and inflammation via the suppression of NFκB [11]. Exercise-induced ROS generation [5] and NFκB-mediated myocardial inflammation [4] are two key pathological triggers we recently demonstrated in ACM.
In addition to either ARBs' efficacy in preventing cardiac dysfunction and fibrosis, we would be remiss to not address the tantalizing outcomes of Pdh and Pdk4 expression in Val-treated Dsg2 mut/mut mice. Pyruvate dehydrogenase catalyzes pyruvate and NAD+ into acetyl-CoA and NADH, the latter of which acts as a reducing agent for the mitochondrial Thioredoxin-2/Peroxiredoxin-3 pathway, a pathway we have shown is severely reduced in ACM myocardial samples [5]. Thus, it appears Valsartan not only inadvertently increases reducing agents (i.e., NADH) to combat ROS levels but also elevates acetyl-CoA levels and thus energy production through the TCA cycle. This is a cycle that has additionally been shown to be down-regulated in ACM myocytes [2]. Lastly, PDK4 inhibits PDH via phosphorylation, thereby contributing to the regulation of fatty-acid oxidation (FAO) via the reduction of glucose metabolism. Considering heart failure (HF) is common in ACM patients, it may seem counterintuitive that Pdk4 expression was significantly downregulated in Val-treated Dsg2 mut/mut mice. Heart failure is a disease state that utilizes glucose metabolism over FAO, which produces far less ATP. Yet, recent work demonstrated that altered cellular metabolism, from glucose metabolism to the TCA cycle, is mediated by the reduction in PDK4 levels [12,13]. That said, Sun Y et al. showed PDK4 binds to the apoptosis-inducing factor (AIF), a mitochondrial protein that we recently showed contributes to cell death in ACM [5].
Although HF shifts the heart to a less energy-efficient metabolic state, new advancements have indicated that increasing glucose utilization in cardiomyocytes is associated with increased cardiac function [12,13]. In conclusion, providers should consider moving alongside antiarrhythmics in ACM, such as the administration of Valsartan as this ARB demonstrates additional metabolic therapeutic effects to increase substrate utilization even in diseased states (i.e., HF) [12,13].

Study Approval
All experiments conformed to the Guide for the Care and Use of Laboratory Animals from the National Institute of Health (NIH publication no. 85-23, revised 1996). Animal study protocol was approved by the Johns Hopkins University Animal Care and Use Committee (Protocol Code: MO19M94; Date of Approval: 26 March 2019).

In Vivo Drug Treatment
Two mouse lines were utilized in this study: Wild-type (WT) controls and homozygous Desmoglein-2 mutant mice (Dsg2 mut/mut ). All mouse lines are of a C57BL/6J background and were bred in-house via heterozygous x heterozygous pairing (i.e., Dsg2 mut/+ x Dsg2 mut/+ ) in order to ensure all mice were age-and litter-matched, with n≥5 mice/cohort/treatment [5]. As previously described, [3] mice with the targeted allele were mated with mice harboring ubiquitous expression of CMV-Cre to remove the fourth and fifth exons of Dsg2 (Dsg2 mut/mut ), which results in the loss of exons 4 and 5 due to a frameshift mutation and premature termination of translation. [3] This led to germline deletion of exons four and five, thus no longer requiring additional Cre-recombinase.

Cardiac Function and Histopathology
Disease functional characterization was performed at 8 and 16 weeks (8 W and 16 W, respectively) of age. Specifically, echocardiography, electrocardiography, and blood pressure plethysmography were obtained at 8 W and 16 W, and histopathology, PPAR protein levels, and qPCR expression were obtained at 16 W.
Echocardiography: A 2100 Vevo Visualsonic was used to assess cardiac function. An ultrahigh-frequency linear array microscan transducer (40 MHz; acquired at a sweep speed of 200 mm/s,) was utilized to obtain short-axis, m-mode and parasternal, long-axis, B-mode images. All images at the level of the papillary muscles were obtained and analyzed following the American Society of Echocardiography guidelines for animals, as previously described [5]. Each mouse had 3-5 measurements obtained for each echocardiographic parameter and then were averaged for statistical evaluation, as previously described [3]. Additionally, all mice were evaluated for the presence of hypokinesia, dyskinesia, and aneurysms at the study endpoint; these functional abnormalities and corresponding data are included in Table 1.
Blood pressure plethysmography (BPP): Non-invasive BPP measurements were obtained via the ADInstruments Tail-cuff BPP System. Specifically, non-anesthetized mice (a) were led into a rodent restrainer (Cat. No. MLA5016); (b) using the slide piston, each mouse was adjusted in order to maximize tail length for placement in the BPP tail cuff holder (Cat. No. MLA5030); (c) a blood pressure cuff (Cat. No. MLT125/m) was then placed around the base of the tail. All BPP measurements were conducted in a 37 • C designated quiet area (JHU Physiology Suite). Prior to BPP measurements, (d) mice were acclimated to the ADInstruments Tail-cuff BPP System for 15mins. Following acclimation, (e) a total of n = 6 BPP measurements were recorded. The first n = 3 measurements were deemed 'acclimation cycles' and discarded, whereas the last n = 3 measurements were averaged for systolic (SBP), diastolic (DBP), and mean BP (MBP) recordings.
Histopathology: Following endpoint functional analyses, mice were euthanized via cervical dislocation, their hearts were extracted and placed in 1X PBS, and they were cut via the long axis. One-half of the heart containing the atrium was processed for immunohistochemical analyses via Masson's Trichrome, as previously described [5]. The remaining half was flash-frozen in liquid nitrogen for downstream analyses.