Dr Ikechukwu Martin Ogbu

Gülbin Kurtay

Dr Margaux Badufle

Prof. Dr Frédéric Robert

Carlos Silva Lopez

Yannick Landais
email: yannick.landais@u-bordeaux.fr

Gulbin Kurtay

] Carlos

Silva Lopez

Dr Gulbin Kurtay

Dr Carlos Silva
email: carlos.silva@uvigo.es

Dr Gulbin

PIDA-mediated Oxidative Decarboxylation of Oxamic Acids. The Role of Radical Acidity Enhancement

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Oxamic acids such as 1 are attractive precursors to access urethanes via isocyanates (Scheme 1a), [1] and for the synthesis of heteroaromatic amides through the Minisci reaction. [2] Inter-and intramolecular additions of oxamic acids to olefins were also reported to provide a broad range of nitrogenated systems. [3] The decarboxylation of these acids was originally achieved by organometallic catalysis using silver and copper salts [1a,2] in the presence of an oxidant in a stoichiometric amount, but more recently photocatalytic [1b-c,4] and electrochemical [5] means have emerged as environmentally more benign solutions. In all cases, the decarboxylation proceeds through a carbamoyl radical intermediate. Our laboratory has shown that the decarboxylation of oxamic acids occurs efficiently under visible light irradiation using a stoichiometric amount of a hypervalent iodine reagent such as BI-OAc (Scheme 1a). [1b,4c] Other I(III) reagents, like BI-OH, BI-OMe, PhI(OAc)2 (PIDA), and PhI(TFA)2 (PIFA), in contrast, were shown to be much less efficient. The photocatalyzed process in Scheme 1a is believed to proceed through the quenching of the photocatalyst in its excited state by an hypoiodite intermediate resulting from ligand exchange at the BI-OAc iodine center. [1b] This then leads to the generation of a carbamoyl radical, which is further oxidized into the desired isocyanate and eventually trapped by the alcohol to give 2. Scheme 1. Oxidative decarboxylation of oxamic acids under photocatalytic and thermal conditions.

Wang and co-workers disclosed a hypervalent iodine-mediated intramolecular Heck-type process involving oxamic acids such as 3 to afford 2-quinolinones 4 (Scheme 1b). [6] The reaction was believed to proceed in this case through a macrocyclic iodine(III) trimer I, the homolysis of which leading to the decarboxylation and the formation of 4. Trimer I was however not isolated and its occurrence was solely based on the characterization through X-ray diffraction studies of a similar trimer formed during the oxidation of a iodobenzene-decorated amino-acid with dimethyl dioxirane. [7] Recent investigations in our laboratory showed that urethanes (i.e. 2) were also accessible through decarboxylation of oxamic acids in the presence of PIDA and an alcohol, but under thermal conditions (vide infra) (Scheme 1c). Our efforts to isolate or unambiguously characterize an hypoiodite, as in the photocatalyzed conditions (Scheme 1a), however met with failure, suggesting a different pathway using PIDA under thermal conditions. It thus appears that the nature of the intermediates formed during the oxidative decarboxylation of oxamic acids in the presence of I(III) reagents as well as the complete reaction pathway to form urethanes remains speculative. In addition, the difference of reactivity between I(III) reagents in these processes, as illustrated by the contrasting behavior of PIDA and BI-OAc, respectively under thermal and photocatalytic conditions, is also unexplained to date. We thus report here on a combined experimental and theoretical study on the oxidative decarboxylation reaction of oxamic acids 5 in the presence of PIDA and alcohols, leading to the formation of urethanes 6 (Scheme 1c). This work provides a complete picture of the process, highlighting the previously overlooked role of the acidity of the NH proton on the carbamoyl radical intermediate, and rationalizes the low reactivity of BI-OAc, as compared to PIDA under thermal conditions.

Results and Discussion

The PIDA-mediated oxidative decarboxylation of oxamic acids into urethanes in the presence of alcohols was thus optimized using oxamic acid 5a as a model compound and ethanol, as summarized in Table 1. At 60°C, in DCE as a solvent, the expected urethane 6a was obtained with 88% conversion after 12h (entry 1). Decreasing the reaction time to 6h still delivered the urethane in high yield (entry 2). PIFA showed reduced efficiency with respect to PIDA, but still provided useful yields (entry 3), BI-OAc or BI-OH, however, resulted in very low to trace amounts of 6a (entry 4-5). This is in staggering contrast with the high yield (80%, vide infra) observed with BI-OAc in the same reaction run under visible light irradiation using 4-CzIPN as a photocatalyst (Scheme 1a). [1b] The hypervalent iodine was shown to be essential for the reaction as no urethane was observed in its absence (entry 6). At 40°C, 75% of urethane was observed, (entry 7), while at room temperature (23°C), the expected urethane was not detected after 12h (entry 8), further highlighting the temperature effect in this process. However, after a prolonged reaction time of five days, the product was formed in satisfying amount (79%), indicating that the reaction still proceeded at room temperature but with low kinetics (entry 9). Using 1.1 eq. of PIDA, 82% conversion was observed after 6h (entry 10), while the reaction did not progress using catalytic amounts (entry 11). Using half equivalent of PIDA led to 50% yield further indicating the need for a stoichiometric amount of PIDA relative to 5a (entry 12). Acid catalysis using BF3-OEt2 inhibited the reaction (entry 13). Interestingly, unlike the photocatalyzed protocol, only proceeding efficiently in DCE or DCM as solvents, the PIDA-mediated procedure progressed smoothly with other organic solvents including DMF and CH3CN (entry 14-15).

Table 1. Optimization of the PIDA-mediated synthesis of urethane 6 from oxamic acid 5a.

Entry [a] Oxidant Time (h) T(°C) Solvent Yield [%] [b] 1 [c] PIDA 6 60 DCE 82 (74) 11 [d] PIDA 12 60 DCE Trace 12 [e] PIDA 12 60 DCE 50 13 [f] PIDA DMF 85 [a] Unless otherwise stated, all reactions were performed with 5 (0.5 mmol), EtOH (3.0 eq.), oxidant (1.5 eq.), solvent (1.0 mL), in a sealable test tube. The heating was done in an oil bath. [b] Yields of 6a were determined by 1 H NMR with 1,3,5trimethylbenzene as an external standard; isolated yields of 6a inside brackets. [c] Reaction using 1.1 eq. of PIDA. [d] Reaction using 10 mol% of PIDA. [e] Reaction using 50 mol% of PIDA. [f] Reaction using 1 eq. of BF3-OEt2.

A series of urethanes 6b-q was then prepared based on the optimized conditions above (entry 1, Table 1), and the scope summarized in Scheme 2. Isolated yields under photochemical conditions reported earlier (using 4-CzIPN as a photocatalyst and BIOAc as an oxidant) were added for comparison. [1b] As indicated, yields under thermal conditions using PIDA compare well with those using photochemical conditions. For instance, with methoxysubstituted benzylic oxamic acid 5i, thermal conditions led to urethane 6i in moderate yield, while photochemical conditions only provided anisaldehyde, resulting from benzylic oxidation without traces of 6i. More generally, all benzylic systems led to satisfying yields using PIDA. Electron-rich and poor arenes 6b, 6e-h were found to be compatible with the reaction conditions, leading to similar yields. Thiophene and furane based oxamic acids afforded the desired urethanes 6j-l in satisfying yields. Bis-oxamic acids were also converted into bis-urethanes 6n-q in moderate to good yields using both conditions.

In order to further understand the reasons for the difference in reactivity between I(III) reagents (entry 1 vs entry 4-5, Table 1) and to determine the reaction pathway of the process, we initially resorted to DFT simulations using 5 (R=Me) as an oxamic model precursor (Scheme 3). First, and to explore the possible pathways for the oxidative decarboxylation of oxamic acids via hypervalent iodine, we assumed that the initial oxamate-acetate anion exchange at the iodine coordination sphere could occur in at least 3 plausible ways: (1) a single oxamic acid molecule could displace the two acetate units in PIDA to form A, as suggested in earlier work, [6,7] (2) a single oxamic acid molecule may displace only one acetate fragment (i.e. B) and (3) two oxamic acid units can displace both acetate fragments (i.e. C).

Table 1. Optimization of the PIDA-mediated synthesis of urethane 6 from oxamic acid 5a.

Scheme 2. Scope of the PIDA-mediated urethane synthesis using oxamic acids 5a-q. Isolated yields of urethanes 6a-q, under thermal conditions. a Under brackets are given isolated yields under photochemical conditions (4-CzIPN, BIOAc, blue LED, RT). [1b] The competition among these three modes of initiation are governed by two basic features, i.e. the acid-base equilibria between oxamic acid and acetic acid in the reaction medium and the kinetic viability and stability of the intermediates generated upon subsequent fragmentation of the complex formed in each alternative.

Scheme 3. Iodine-ligand exchange in PIDA using oxamic acid 5 (R = Me) (PBE0/Def2SVP relative free energies in kcal/mol, using methanol as solvent through the PCM formalism).

As shown in Scheme 3, the chelate A formation by dual substitution of the acetate ligands with a single unit of oxamic acid is disfavored, which contrasts with the known formation of the corresponding 5-membered ring from -amino acids. [6,7] Subsequent fragmentation to yield an isocyanate intermediate, however, is kinetically inexpensive (once the chelate A is formed) with an energy barrier of only 15.2 kcal/mol (Scheme 4). The combination of the energetic cost of both steps (31.8 kcal/mol) however suggests that this reaction would not be operative under the thermal conditions used in our experiments. We hypothesized that such a mechanism to decarboxylate oxamic acids and other similar derivatives may only operate if relatively strong bases or acids [8] are added to the reaction medium so that they facilitate the chelate formation, which contrasts with the optimized conditions in Table 1.

Scheme 4. Reaction path from oxamic acid to the observed carbamate via a single chelating substitution at PIDA (PBE0/Def2SVP relative free energies in kcal/mol, using methanol as solvent).

Single acetate substitution by oxamic acid to afford complex B was therefore considered. In this case the exchange reaction is thermodynamically favored by about 6 kcal/mol, a natural consequence of not requiring the deprotonation at the nitrogen site and in good agreement with the higher acidity of oxamic vs. acetic acid. We were able to locate a concerted transition state on an energy plateau for the direct fragmentation of this complex into four species: carbon dioxide, iodobenzene, acetoxyl carbamoyl radicals (Scheme 5). The barrier for this step is 27.2 kcal/mol, significantly lower than the alternative for the chelate A considered above. The fragmentation process is also slightly favored thermodynamically, compared to the reacting complex N (by -2.7 kcal/mol). For this reaction mode, however, we could not find any evolution of the fragmentation intermediates that would afford the observed carbamate with low or moderate barriers likely due to the unavoidable formation of oxygen-centered radical species (Scheme 5). From the carbamoyl intermediate (INT1) we explored a stepwise mechanism involving hydrogen atom abstraction from methanol, both at the C and the O atoms to yield N-methyl formamide which would subsequently be attacked by the formed radical (either methoxyl or hydroxymethyl, see Scheme 5). Both paths appear as dead ends due to the eventual formation of oxygen centered radicals, which are quite unstable and high in energy (INT3B and INT3B'). These results are in good agreement with the experimental results, since no C-C bond formation was ever observed in this chemistry, as the evolution of INT3B' would imply. A concerted addition of methanol onto the carbamoyl intermediate via TS5B, was also considered in order to avoid oxygen centered radical intermediates. This alternative was however only marginally less demanding than the stepwise pathway (35.6 kcal/mol) and thus, kinetically unfavourable. A third route involving the barrierless recombination of the two radicals formed at the starting fragmentation yields O-acyl carbamate INT2B'' which, through a retroene reaction, would yield isocyanate and acetic acid. This latter pathway is energetically feasible although it implies the initial recombination of two transient radical species that should be present in very small concentrations under the reaction conditions. Thus, an energetically similar pathway not involving such unlikely event would be kinetically more favourable.

Scheme 5. Reaction path from oxamic acid to the observed carbamate via a single substitution at PIDA (PBE0/Def2SVP relative free energies in kcal/mol, using methanol as solvent).

Finally, we considered the possibility of a double oxamate-acetate exchange at the hypervalent iodine center (i.e. C, Scheme 3). The formation of the dioxamate C derivative is indeed quite favourable (-16.3 kcal/mol) and, despite this stability, the subsequent fragmentation transition state is affordable (with a barrier of only 7.6 kcal/mol, Scheme 6). We strived for the location of a concerted transition state in which both oxamic units decarboxylate, therefore yielding two carbamoyl radicals and two molecules of CO2 in a single step. Such transition state could not be located. It is however worth noticing that once the first oxamate is fragmented, it leaves behind a loosely bound mono-oxamate radical complex INT1C, which suffers decarboxylation in a barrierless manner (Scheme 6).

In essence, a single kinetically relevant transition state is responsible for the fragmentation of both units of the oxamic acid, although the actual decarboxylation events for each of the oxamate units are extremely asynchronous. [9] The first decarboxylation is roughly thermoneutral, but once the second one is considered, the overall process is exergonic by about 26 kcal/mol. This, therefore, seems to be the most plausible reaction mode for the title reaction, but the question still remains on how, and with what energy cost, are the two units of carbamoyl radical INT1 transformed into the observed isocyanate? Scheme 6. Reaction path from oxamic acid to carbamoyl intermediates via a double substitution at PIDA (PBE0/Def2SVP relative free energies in kcal/mol, using methanol as solvent).

In this last alternative, two carbamoyl radicals INT1 are formed for each molecule of PIDA employed and, since the evolution of these radicals via reaction with alcohol has already been discarded due to the formation of very unstable intermediates, the only option left is the intervention of a second molecule of PIDA in agreement with the stoichiometry of the reaction (Table 1, entries 9-12). We considered the displacement of acetate by these radical species, but the lack of nucleophilicity and their inability to neutralize the positive charge on iodine results in unfavourable energetics (Supporting information). The displacement of acetate from PIDA therefore seemed to require deprotonation at the N site of the carbamoyl units. This deprotonation was responsible for high energy barriers and unfavorable thermodynamics in the formation of the chelate A (Scheme 3). Interestingly, however, radical enhanced deprotonation (RED-shift) [10] has been described for related radicals such as carboxyl (HO . C(=O)) and hydroperoxyl radicals (HOO . ) for instance, which are strong Brønsted-Lowry acids. These transient radical species exhibit enhanced acidity as compared with the saturated valence analogue (pka 3.85 and 6.69 for HOC(=O)H respectively). A similar enhanced acidity was calculated for INT1 with a pka of 5.45 as compared to the corresponding formamide HC(=O)NHMe (See supporting information). [11] This effect may explain why the single acetate-carbamoyl anion displacement is only mildly endergonic (6.4 kcal/mol in Scheme 7 vs 16.6 kcal/mol for the chelate A in Scheme 3). Additionally, the formed intermediate INT2C is rather unstable and fragments with a barrier of less than 5 kcal/mol to furnish methyl isocyanate and a periodinane monoacetate radical, INT3C (Scheme 7). A termination sequence involving the radical collapse of the remaining unit of carbamoyl radical and the monoacetate occurs without barrier and forms a second isocyanate molecule. From this point, two additions of alcohol onto the recently formed isocyanate molecules yield the observed carbamates.

Scheme DFT calculations on the PIDA-mediated oxidative decarboxylation of oxamic acids under thermal conditions (PBE0/Def2SVP relative free energies in kcal/mol, using methanol as solvent through the PCM formalism).

A complete picture of the PIDA-mediated decarboxylative oxidation of oxamic acid may thus be proposed as depicted in Figure 1 below. This mechanism involves the formation of a carbamoyl radical intermediate INT1, in contrast with the early work of Kita and co-workers [6a] on the I(III)-mediated oxidative fragmentation of -amino acids where no radical species could be observed through ESR. The mechanism was believed to proceed instead through the fragmentation of a 5-membered ring chelate closely related to high energy intermediate A in Scheme 3. The reaction path hereby proposed is however compatible with a number of observations on this chemistry. For instance, with this profile in hand, we could also explain why PIFA and BI-OAc are not as efficient for this chemistry. PIFA features a less basic anion that hampers the thermodynamics of the initial oxamic exchange, but it can still operate through the most competitive double substitution mechanism, hence the moderate reduction in yield. BI-OAc, however, can only operate through the single substitution path since only one acetate unit is available for ligand exchange (the benzoate I-O bond is unlikely to be released without a high energy penalty).

As we have seen with PIDA in this work, the single substitution alternative is far less competitive than the double ligand exchange, hence the deleterious effect on the kinetics the reaction yield (details on these two alternatives can be found in the Supporting information). Another consequence of the hereby proposed reaction mechanism is that N,N-disubstituted oxamic acid derivatives should not react with PIDA to form carbamates (vide infra). Our mechanism anticipates the need for at least one proton on nitrogen for the reaction to be operative. For this reason, and to verify the plausibility of this proposal, we decided to run a series of experiments using 5a as a model oxamic acid (Scheme 8). The radical nature of the reaction was first established using TEMPO as a radical trapping agent. The presence of TEMPO totally quenched the formation of 6a and traces of carbamoyl radical-TEMPO adduct 7 were isolated from the reaction mixture as confirmed by HRMS. [START_REF] De Mico | The low yield of 7 may result from the competitive reaction between PIDA and TEMPO[END_REF] The treatment of 5a using 1.5 eq. of PIDA under above reaction conditions, but in the absence of alcohol, confirmed the in-situ generation of isocyanate 8. Interestingly, while the occurrence of a carbamoyl radical intermediate INT1 (Scheme 6) was assessed by the detection of 7, when the PIDA-mediated oxidation was repeated in the presence of 4-methyl-quinoline (not shown), no trace of the amide resulting from the Minisci addition [4c] was observed (Figure S1-2), [START_REF]Small amount of isocyanate 8 was formed during the process[END_REF] which may be ascribed to the rapid trapping of carbamoyl radical INT1 to form INT2C, which readily suffers fragmentation into INT3C and methyl isocyanate (Scheme 7).

To further interrogate the reaction pathway of this process, and the possible occurrence of a cationic carbamoyl species (a protonated isocyanate form), the intermolecular addition of 5a to electron rich 1,2-dimethoxybenzene was tested but the expected amide 9 was not observed. Instead, diamide 10 was isolated in 17% yield, again confirming the formation of the carbamoyl radical INT1, which is known to readily dimerize. [5a] These results further show that the oxidative decarboxylation reaction of oxamic acids in the presence of PIDA follows a distinct pathway from that of BI-OAc under photochemical conditions. [1b,4c] Finally, the process was extended to secondary oxamic acid 11, which remained unchanged under the above conditions, pointing out the crucial role of the oxamic NH proton and the radical enhanced acidity effect raised by DFT calculations (Scheme 7).

Scheme 8.

Trapping experiments during PIDA-mediated oxidative decarboxylation of oxamic acids under thermal conditions.

Conclusion

As a summary, experimental studies supported by DFT calculations reveal that the PIDA-mediated decarboxylation of oxamic acids into isocyanates proceeds through a mechanism encompassing several keyfeatures: (1) a double exchange at the iodine center between OAc and oxamic acid ligands; (2) the formation of a carbamoyl radical, detected experimentally; (3) an unprecedented exchange between this radical and an OAc ligand on PIDA facilitated by the increased acidity of the carbamoyl radical NH. [10] Experimental Section General procedure: Oxamic acid 5 (1.0 eq.), ROH (3.0 eq.) and PIDA (1.5 eq.) were charged into an oven-dried sealable test tube equipped with a magnetic stirrer, and dissolved in DCE (0.5 M). The content was degassed and backfilled with argon. The tube was heated to 60°C (oil bath) during 12 h. The reaction mixture was then concentrated under vacuum and the crude purified by flash column chromatography on silica gel (Column: 2 cm x 10 cm, SiO2 x 100: 15 g) using petroleum ether/EtOAc as an eluent (ratio depending on the nature of the substrate) to afford the product.

Figure 1 .

 1 Figure 1. DFT calculations on the PIDA-mediated decarboxylative oxidation of oxamic acids. Complete pathway. (PBE0/Def2SVP relative free energies in kcal/mol, using methanol as solvent. Geometry of the transition state for the starting fragmentation with key bond lengths (in Angstroms)

Acknowledgements

IMO and GK thank the Alex-Ekwueme Federal University Ndufu-Alike Ikwo (AE-FUNAI) and the TUBITAK BIDEB-2219 program (grant N°1059B191900553) respectively for a PhD and a post-doctoral grant. We gratefully acknowledge the ANR (NCO-INNOV, N°20-CE07-0015-01), the University of Bordeaux and the CNRS for financial support. CSL thanks the Minsterio de Ciencia e Innovación (PID2020-115789GB-C22) and Xunta de Galicia (ED431C 2021/41), which partially funded this work. Generous allocation of supercomputer resources by CESGA is also acknowledged.

Keywords

: isocyanate • urethane • hypervalent iodine • DFT • carbamoyl radical • acidity •


Michael J Wallace

Nipun Malhotra

Juan Ignacio

Elio Mariángelo

Tyler L Stevens

Lindsay J Young

Steve Antwi-Boasiako

Danielle Abdallah

Sarah Sumie Takenaka

Omer Cavus

Nathaniel P Murphy

Juan Ignacio Elio Mariángelo

Mei Han

Xianyao Xu

Matteo E Mangoni

Thomas J Hund

Jason D Roberts

Sandor Györke

Peter J Mohler

Mona El Refaey

PhD, MS Mona El Refaey
email: mona.elrefaey@osumc.edu

Impact of stress on cardiac phenotypes in mice harboring an ankyrin-B disease variant

Keywords: ankyrin-B, bradycardia, incomplete penetrance, heart rate variability, fibrosis, arrhythmia, variant, heart failure, stress, cardiovascular disease

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Ankyrin-B (AnkB), encoded by ANK2, was first identified to play a role in the nervous system via maintenance of pre-myelinated axons [START_REF] Scotland | Nervous system defects of AnkyrinB (-/-) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kD AnkyrinB in premyelinated axons[END_REF], and subsequently determined to impact cardiac function. AnkB regulates the expression and targeting of key membrane, cytoskeletal, and regulatory proteins, including the Na + /Ca 2+ exchanger (NCX), Na + /K + -ATPase (NKA), voltage-gated calcium channel (Cav1.3), protein phosphatase 2 A (PP2A) and β-spectrin [START_REF] Cunha | Targeting and stability of Na/Ca exchanger 1 in cardiomyocytes requires direct interaction with the membrane adaptor ankyrin-B[END_REF][START_REF] Koenig | The evolving role of ankyrin-B in cardiovascular disease[END_REF]. ANK2 encodes multiple isoforms and is subject to transcriptional regulation [START_REF] Cunha | Exon organization and novel alternative splicing of the human ANK2 gene: implications for cardiac function and human cardiac disease[END_REF].

AnkB dysfunction is associated with diverse human cardiac phenotypes, including QT prolongation, arrhythmogenic cardiomyopathy (ACM), sinus node dysfunction, and atrial fibrillation. Rare ANK2 variants may also function as modifiers of wall thickness in hypertrophic cardiomyopathy [START_REF] Lopes | Novel genotype-phenotype associations demonstrated by high-throughput sequencing in patients with hypertrophic cardiomyopathy[END_REF]. The broad spectrum of AnkB molecular partners in the heart likely accounts for the corresponding broad range of cardiac phenotypes associated with ANK2 dysfunction. The constellation of clinical features observed with ANK2 dysfunction has been termed 'ankyrin-B syndrome' [START_REF] Curran | Coordinating electrical activity of the heart: ankyrin polypeptides in human cardiac disease[END_REF][START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF][START_REF] Choi | Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels[END_REF][START_REF] Lin | Applying High-Resolution Variant Classification to Cardiac Arrhythmogenic 29 Gene Testing in a Demographically Diverse Cohort of Sudden Unexplained Deaths[END_REF][START_REF] Hedley | The genetic basis of long QT and short QT syndromes: a mutation update[END_REF].

The human AnkBp.Glu1458Gly variant is associated with arrhythmias and structural remodeling [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF]. However, ankyrin-B syndrome displays incomplete penetrance, suggesting that AnkB variants alone may not be sufficient to directly cause arrhythmia and/or structural phenotypes. Mouse gene knockout models may not replicate human molecular phenotypes (e.g. in case of dominant-negative variants). Therefore, we engineered a new animal model harboring the AnkB variant p.E1458G. Here, we illustrate two points: 1, single ankyrin-B variants are sufficient to cause cardiac phenotypes in six-month old AnkBp.E1458G +/+ mice, and 2, acute and/or chronic stress augments the genesis of cardiac disease phenotypes in both young and old mice carrying the human variant. Our findings support a role of single ANK2 variants in promoting mild cardiac phenotypes in vivo. However, severe in vivo phenotypes require secondary stressors including aging and adrenergic-stress.

Results

Generation of a novel humanized knock-in mouse model

The AnkBp.E1458G variant (Figure 1A) is associated with ankyrin-B syndrome [START_REF] Koenig | The evolving role of ankyrin-B in cardiovascular disease[END_REF] and ACM [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF]. To test if this variant is sufficient to induce cardiac phenotypes in vivo, we generated a homozygous knock-in (KI) model (Figure 1B-C) harboring this single amino acid substitution. Cas9 was used to introduce a double strand chromosome break near codon 1371 in mouse (corresponds to 1458 in human sequence, ENSMUST00000182078.9). A single stranded oligonucleotide donor was used to change E1371 (E1458) to G1371 (G1458) and multiple silent coding changes were also introduced to block the Cas9 targets. Sanger sequencing chromatograms were used to confirm the substitution of glutamic acid (E) by glycine (G) in the KI mouse (Figure 1B-C). AnkBp.E1458G +/+ mice were born at normal Mendelian ratios, 29% control mice (40/137), 46% heterozygous mice (63/137) and 25% homozygous mice (34/137). Ank2 mRNA expression levels were not changed in hearts of AnkBp.E1458G +/+ mice compared to control littermates (Figure 1D). Canonical AnkB protein levels were not significantly different between AnkBp.E1458G +/+ and control littermate hearts (Figure 1E andF).

Young AnkBp.E1458G +/+ mice do not display cardiac phenotypes Three-month old AnkBp.E1458G +/+ homozygous mice did not display structural phenotypes. Specifically, three-month old AnkBp.E1458G +/+ mice did not display changes in ejection fraction or fractional shortening (Figure 2A, Suppl. Figure 1A, G andH). Moreover, AnkBp.E1458G +/+ mice did not exhibit electrocardiogram alterations or arrhythmia phenotypes at three months of age (Suppl. Figure 2).

Aged AnkBp.E1458G +/+ mice display structural remodeling and reduced cardiac function

AnkBp.E1458G +/+ mice showed a significant reduction in ejection fraction and fractional shortening at ~six months of age (Figure 2B-D, Suppl. Figure 1B and Suppl. Table 1). Six-month old AnkBp.E1458G +/+ mice also exhibited a trend towards an increased LVID (p=0.0694, Suppl. Figure 1C) and decreased LVPW during systole (p=0.0971, Suppl. Figure 1D) that did not reach statistical significance. No significant changes were noted in LVID or LVPW during diastole (Suppl. Figure 1E-F). While Ank2-cKO mice showed left ventricular dilation and increased body weight relative to tibial length (7), these findings were not observed in the AnkBp.E1458G +/+ mice in this study (Figure 2E-G). Notably, six-month old AnkBp.E1458G +/+ mice showed widespread cardiac fibrosis at baseline compared with control mice (Figure 2F andH). In summary, aged AnkBp.E1458G +/+ mice developed structural remodeling characterized by cardiac fibrosis and reduced cardiac function in the absence of stress. No significant changes were noted in the cardiac remodeling markers including β-myosin heavy chain (Myh7), atrial natriuretic peptide (Nppa), brain natriuretic peptide (Nppb), Collagen type I alpha 1 chain (Col1a1) and Tissue inhibitor matrix metalloproteinase 1 (Timp1) in the AnkBp.E1458G +/+ mice at ~ six months of age (Suppl. Figure 3). 3A andB; p=0.03). Accordingly, the RR interval was significantly prolonged in the AnkBp.E1458G +/+ mice compared to the control littermates (mean RR =0.0919 ± 0.0024 s in control mice vs. mean RR in AnkBp.E1458G +/+ mice = 0.1035 ± 0.0040 s; Figure 3F; p=0.04). No significant changes were noted in the P wave duration, PR interval or the QRS duration (Figure 3C-E and Suppl. Table 2). AnkBp.E1458G +/+ mice showed a statistically significant prolongation in the QT-interval (mean= 0.0206 ± 0.0007 s in control mice vs. mean= 0.0231 ± 0.0006 s, p=0.02). However, the corrected QT interval (Mitchell et al. (11)) trended toward prolongation in the AnkBp.E1458G +/+ mice, but did not reach statistical significance (mean= 0.0216 ± 0.0006 s in control mice vs. mean= 0.0228 ± 0.0007 s, p=0.25;Figure 3G andH). There is a debate on whether the QT interval is affected by heart rate in small animals [START_REF] Mulla | Incorrectly corrected? QT interval analysis in rats and mice[END_REF][START_REF] Roussel | The Complex QT/RR Relationship in Mice[END_REF][START_REF] Kmecova | Heart rate correction of the QT duration in rats[END_REF] and therefore, we measured both intervals and displayed QTc according to Mitchell et al [START_REF] Mitchell | Measurement of heart rate and Q-T interval in the conscious mouse[END_REF].

Aged AnkBp.E1458G

Abnormal high and low-frequency components of heart rate variability (HRV) may suggest a dysfunction in autonomic regulation or an intrinsic sinus node dysfunction. Specifically, the high frequency (HF) component is modulated by the parasympathetic nervous system, while the low frequency (LF) reflects the activity of both parasympathetic and sympathetic nervous system [START_REF] Billman | The LF/HF ratio does not accurately measure cardiac sympatho-vagal balance[END_REF]. The high-and lowfrequency spectral components of HRV were significantly increased in the AnkBp.E1458G +/+ mice versus control littermates at rest (Figure 4A (p<0.01) and B (p=0.02)) in line with mild bradycardia observed in AnkBp.E1458G +/+ mice. Notably, LF/HF ratio was significantly reduced in the AnkBp.E1458G +/+ mice (Figure 4C p=0.03). Analyzing the RR-interval of the control mice vs. the AnkBp.E1458G +/+ mice over a 14 min interval confirmed and displayed both prolongation and variability in the aged mice harboring the single amino acid substitution, AnkBp.E1458G. Analysis of RR and standard deviation of RR (SDRR) intervals displayed a significant increase in both parameters in the AnkBp.E1458G +/+ vs. the control mice (Figure 4D-E).

Representative traces from both genotypes over 1 min interval are displayed in Figure 4F.

AnkBp.E1458G +/+ mice exhibit arrhythmia in response to adrenergic challenge

Previous studies using Ank2-cKO mice noted polymorphic arrhythmia events following adrenergic stimulation [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF]. Young mice harboring the human variant displayed an increase in the number of ventricular arrhythmic events (control, mean~0.7 events vs. AnkBp.E1458G +/+ mice, mean ~18 events) (Figure 5A-D, p=0.0325) when challenged with epinephrine. Moreover, six-month old mice also showed a significant increase in the number of ventricular arrhythmia events compared to their control littermates following epinephrine (control, mean ~14.5 ± 6.14 events and AnkBp.E1458G mice, mean ~35.8 ± 8.66 events) (Figure 6A, p=0.04). Finally, when challenged with a stress protocol consisting of combined epinephrine (2mg/kg) and caffeine (120mg/kg), the six-month old AnkBp.E1458G +/+ mice displayed a significant increase in the number of ventricular arrhythmic events (control, mean ~18.3 ± 6.34 events and AnkBp.E1458G mice, mean ~116.8 ± 41.73 events) (Figure 6B, p=0.04). Notably, the number and duration of sustained episodes of ventricular tachycardia (VT) were significantly higher and prolonged in the AnkBp.E1458G +/+ mice compared with control littermates (Figure 6C-D, p<0.01 and p=0.03). The number of non-sustained ventricular tachycardia (NSVT) episodes trended upward in AnkBp.E1458 +/+ mice (Figure 6E, p=0.14). Representative recordings denoting episodes of VT and NSVT are included in (Figure 6F-H). Taken together, AnkBp.E1458G +/+ mice showed arrhythmic events following adrenergic challenge or acute stressor at an early age. These events became more severe and sustained in adult animals at six months of age.

Abnormal calcium handling may lead to cardiac electrical abnormalities either through spontaneous calcium waves that cause delayed afterdepolarizations or through calcium transient alternans, that provide a substrate for reentrant mechanisms [START_REF] Laurita | Cellular mechanisms of arrhythmogenic cardiac alternans[END_REF]. In this study, we investigated calcium handling in cardiac myocytes derived from AnkBp.E1458G +/+ mice and control littermates in the presence (100 nM and 500 nM) and absence of isoproterenol (ISO). As expected, ISO significantly increased the amplitude and rate of decay of calcium transients (Figure 7A-C). We observed that the amplitude and rate of decay of calcium transients were similar between AnkBp.E1458G +/+ and control myocytes ± ISO. Additionally, the frequency of spontaneous calcium waves was comparable between the two groups (Figure 7D and E). Notably, AnkBp.E1458G +/+ myocytes displayed a higher predisposition to calcium transient alternans than WT myocytes (Figure 7F-H). In line with previous studies [START_REF] Florea | Regulation of cardiac alternans by betaadrenergic signaling pathways[END_REF][START_REF] Hammer | Cardiac myocyte alternans in intact heart: Influence of cell-cell coupling and beta-adrenergic stimulation[END_REF], ISO tended to decrease the likelihood of alternans in WT myocytes.

However, in AnkBp.E1458G +/+ myocytes, ISO increased the predisposition to alternans (Figure 7G-H). Overall, these findings illustrate calcium handling dysfunction in AnkBp.E1458G +/+ myocytes.

AnkBp.E1458G +/+ mice develop severe cardiac phenotypes in response to chronic cardiac pressure overload

We hypothesized that young AnkBp.E1458G homozygous mice would develop an accelerated structural phenotype in response to chronic cardiac stress. Therefore, a model of pressure overload, transverse aortic constriction (TAC), was performed in young AnkBp.E1458G +/+ mice. AnkBp.E1458G +/+ mice exhibited a significant reduction in contractility or ejection fraction and fractional shortening at eight and twelve weeks post TAC compared to control littermates (Figure 8A andB). Moreover, AnkBp.E1458G +/+ mice did not display significant differences in the LVID during diastole or systole post TAC (Figure 8C-D). Furthermore, AnkBp.E1458G +/+ hearts displayed widespread cardiac fibrosis in the homozygous hearts post TAC (Figure 8E and F) compared with control mice. Comparing the relative expression of cardiac remodeling markers between the young mice (~2-3months of age) and mice post TAC, no significant changes were noted in Myh7. We observed a significant increase in Nppa relative expression in the control and AnkBp.E1458G +/+ hearts post TAC vs. young control hearts (p=0.026 and p=0.025) and vs. young AnkBp.E1458G +/+ hearts (p=0.032 and p=0.0036). Nppb relative expression was upregulated in the AnkBp.E1458G +/+ hearts post TAC compared to the young controls (p=0.0168) and young AnkBp.E1458G +/+ hearts (p=0.0125). Col1a1 relative expression was upregulated in the AnkBp.E1458G +/+ hearts post TAC compared to the young AnkBp.E1458G +/+ (p=0.0467). Finally, Timp1 was significantly upregulated in the control (p=0.0382) and AnkBp.E1458G +/+ (p=0.0199) hearts post TAC compared to young AnkBp.E1458G +/+ mice (Suppl. Figure 4).

Expression and localization of AnkB membrane partners is altered in

AnkBp.E1458G +/+ hearts

We investigated the expression of AnkB and AnkB-key binding partners, including NCX and NKA, in our AnkBp.E1458G +/+ mouse model at baseline, older age and post TAC. Immunoblotting experiments showed no changes in the normalized expression of AnkB in the AnkBp.E1458G +/+ mice at six months of age (Figure 9A andB). Additionally, Ank2 mRNA levels were not different between the two groups of mice at ~six months of age (Suppl. Figure 5). The expression of both NCX and NKA was not different in the young AnkBp.E1458G +/+ mice at ~2 months of age (Suppl. Figure 6A-D). The expression of NCX was not different between the groups (Figure 9C andD) at ~six months of age. However, NKA protein expression normalized to GAPDH showed a significant decrease in the hearts of the aged AnkBp.E1458G +/+ mice (~30% reduction, p= 0.0023) (Figure 9E andF). Ankyrin-G expression was unchanged between the two groups of mice (Figure 9G and Suppl. Figure 7). Notably, NCX expression remained unchanged and the NKA expression was significantly reduced in the AnkBp.E1458G +/+ mice post TAC compared to the control mice (Suppl. Figure 6E-H). Finally, NKA protein was bound to the AnkB Ig in the presence of the human variant (Suppl. Figure 8).

AnkB is a cytoskeletal protein that anchors and regulates the organization and localization of both NKA and NCX in a macromolecular complex. We observed no significant changes in the localization of NKA and NCX in AnkBp.E1458G +/+ isolated myocytes in relation to ankyrin-B in young mice (Figure 10A-B, E-F). However, immuno-labeling data in the aged AnkBp.E1458G +/+ myocytes demonstrated a reduction in NKA localization at the T-tubules (Figure 10C,G). NCX signal was consistently similar in the aged AnkBp.E1458G +/+ and control myocytes (Figure 10D,H). In summary, six months old AnkBp.E1458G +/+ myocytes display altered NKA expression and localization.

Discussion

The human AnkBp.E1458G variant was initially identified in a large French family with a complex cardiac phenotype, including sinus node dysfunction, ventricular arrhythmia, sudden cardiac death, and atrial fibrillation [START_REF] Curran | Coordinating electrical activity of the heart: ankyrin polypeptides in human cardiac disease[END_REF]. This phenotype, referred to as "AnkB-syndrome", has been attributed to altered Ca 2+ signaling when studied in a full-body haploinsufficient AnkB murine model [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF][START_REF] Scouarnec | Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease[END_REF]. The AnkBp.E1458G variant has also been implicated in other cardiac conditions, such as ACM [START_REF] Lopes | Novel genotype-phenotype associations demonstrated by high-throughput sequencing in patients with hypertrophic cardiomyopathy[END_REF] and as a potential modifier of wall thickness in hypertrophic cardiomyopathy [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF]. Notably, the same AnkBp.E1458G variant was identified by Hertz et al. in children who suffered from sudden unexpected infant death [START_REF] Hertz | Genetic investigations of sudden unexpected deaths in infancy using next-generation sequencing of 100 genes associated with cardiac diseases[END_REF][START_REF] Neubauer | Post-mortem whole-exome analysis in a large sudden infant death syndrome cohort with a focus on cardiovascular and metabolic genetic diseases[END_REF]. In summary, the range of cardiac phenotypes identified to harbor the human AnkBp.E1458G variant are diverse and complex.

Despite numerous reports identifying the AnkBp.E1458G missense variant in cases of cardiac disease, the variant has also been identified in non-diseased populations, raising questions about its pathogenicity and penetrance [START_REF] Lin | Applying High-Resolution Variant Classification to Cardiac Arrhythmogenic 29 Gene Testing in a Demographically Diverse Cohort of Sudden Unexplained Deaths[END_REF][START_REF] Reuter | The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants[END_REF].

Interestingly, Ware and colleagues reported that 49 of 64 published ANK2 variants were either "benign" or "probably benign" [START_REF] Ware | Paralogous annotation of disease-causing variants in long QT syndrome genes[END_REF]. AnkB plays a role in the transport and localization of numerous proteins to the membrane, transverse-tubule, and intercalated disc in cardiomyocytes. Although, the AnkBp.E1458G variant is not located where AnkB associates with key binding partners, the variant is localized close to the site critical for AnkB intramolecular association.

GnomAD data estimates the minor allele frequency of the AnkBp.E1458G AnkB variant to be 0.000535 (https://gnomad.broadinstitute.org/variant/4-114269433-A-G?dataset=gnomad_r2_1). Further, the AnkBp.E1458G variant is significantly more prevalent among individuals with European and Latin ancestry. Therefore, it is more frequent in the general population than the AnkB syndrome phenotype [START_REF] Adler | An International, Multicentered, Evidence-Based Reappraisal of Genes Reported to Cause Congenital Long QT Syndrome[END_REF], consistent with the incomplete penetrance of the AnkB phenotype associated with the AnkBp.E1458G variant. These findings, coupled with the AnkBp.E1458G variant most often being identified in sporadic cases of cardiac disease without clear evidence of familial genotype-phenotype segregation, indicate that the variant must rely on additional genetic and/or environmental factors in order to manifest with a clinical phenotype.

The translational relevance of the haploinsufficient and knockout mice has previously been questioned given the strong mouse phenotype compared to the commonly mild human phenotype, coupled with ANK2 variants implicated in human disease most often being missense [START_REF] Giudicessi | Established Loss-of-Function Variants in ANK2-Encoded Ankyrin-B Rarely Cause a Concerning Cardiac Phenotype in Humans[END_REF]. Recently, developments in gene editing technology have allowed us to study the specific AnkBp.E1458G variant in mice, that may more effectively model the human phenotype. The newly developed KI murine model homozygous for the AnkBp.E1458G variant (Figure 1) provides a new tool and new insights into the pathogenesis and penetrance of the AnkB syndrome.

Notably, aged AnkBp.E1458G +/+ mice displayed bradycardia (Figure 3) and increased HF and LF spectral elements suggesting higher HR variability in mice at a later age while the mice displayed a lower LF/HF ratio (Figure 4). Increase in HF and LF is in line with lower basal heart rate observed in AnkBp.E1458G mice. The increase in HRV seen in the KI mice might suggest an intrinsic sinus node dysfunction associated with this variant that leads to increased heart variability as suggested by a previous report [START_REF] Sosnowski | Heart rate variability. Is it influenced by disturbed sinoatrial node function?[END_REF]. However, we cannot exclude that AnkBp.E1458G may also present with increased activity of the parasympathetic nervous system. Additionally, homozygous mice showed increased vulnerability to arrhythmic events in response to adrenergic challenge as early as ~three months of age post adrenergic stimulation (Figure 5). Aged mice showed propensity for more severe cardiac arrhythmic events, more frequent events, and longer sustained runs of arrhythmic episodes in response to adrenergic and caffeine administration (Figure 6). AnkBp.E1458G +/+ myocytes displayed a higher a predisposition to calcium transient alternans that provides a potential substrate for reentrant mechanisms (Figure 7). TAC surgery, a model for chronic cardiac stress, also induced an accelerated heart failure phenotype in the young AnkBp.E1458G +/+ mice (Figure 8). Overall, stress-inducing studies support our hypothesis that the AnkBp.E1458G variant may be more likely to manifest with a cardiac phenotype in response to environmental stressors and aging. Similar results were reported in a truncated Plakophilin-2 (PKP2) mouse model (p.R735X).

Plakophilin 2 variants are the most common ACM-causing genetic variants, accounting for 23-58% of all ACM cases. However, the ACM phenotype did not manifest in the PKP2p.R735X transgenic mice until exposure to eight weeks of endurance exercise training (another environmental stressor) [START_REF] Cruz | Exercise triggers ARVC phenotype in mice expressing a disease-causing mutated version of human plakophilin-2[END_REF][START_REF] Stevens | Arrhythmogenic Cardiomyopathy: Molecular Insights for Improved Therapeutic Design[END_REF].

Previous studies have shown NKA and AnkB dissociation as a prominent mechanism for myocardial cell death after myocardial ischemia [START_REF] Inserte | Calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion contributes to cell death after myocardial ischemia[END_REF]. Calpainmediated degradation of the NKA anchorage to the membrane cytoskeleton resulted in Na + overload, which had a secondary effect on NCX resulting in secondary Ca 2+ overload within the cell following ischemia [START_REF] Inserte | Calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion contributes to cell death after myocardial ischemia[END_REF]. Moreover, Skogestad et al. recently demonstrated how disruption in the AnkB/NKA interaction resulted in increased Ca 2+ sparks [START_REF] Skogestad | Coupling of the Na+/K+-ATPase to Ankyrin B controls Na+/Ca2+ exchanger activity in cardiomyocytes[END_REF]. Inhibition of NKA raises [Na + ]i, and therefore influences the function of NCX. In order to extrude excess Na + , [Ca 2+ ]i becomes elevated, which can result in altered Ca 2+ cycling. Interestingly, Li et al. identified that inhibition of NKA during stressed conditions can elicit pro-arrhythmic alternans in guinea pig cardiomyocytes [START_REF] Li | Inhibiting Na+/K+ ATPase can impair mitochondrial energetics and induce abnormal Ca2+ cycling and automaticity in guinea pig cardiomyocytes[END_REF]. Increased [Ca 2+ ]i may result in calcium alternans that may translate into conduction block and reentrant arrhythmia via non-uniform refractoriness throughout the electrical signaling pathways (32).

There are limitations to our work. Affected individuals in the setting of AnkBp.E1458G have been in the heterozygous state. Notably, the phenotype of the heterozygous mouse models (usually haploinsufficient) for desmosomal variants are modest (often nothing is observed) and hence desmosomal genetic culprits are also frequently studied in the homozygous state [START_REF] Cerrone | Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm[END_REF]. Although gene dosage may be higher, this mouse model still serves as an effective model for evaluating the potential pathologies associated with this AnkB variant. A pressure overload model has not previously been shown to predispose to ACM and to our knowledge, there is no clinical data to suggest that pressure overload or hypertension exacerbates clinical phenotype in the setting of an ANK2 variant. Conclusions from this study should be extrapolated with caution to other pathogenic human ANK2 variants. Gene delivery and pharmacological therapeutics can both be tested in vivo using this knock-in model. While our studies were performed in vivo, it will be still crucial for expansion of these studies into large animal models. Human induced pluripotent stem cells (iPSCs) would best allow for specific excitation-contraction coupling and further Ca 2+ regulation studies. In summary, while ANK2 variants may be loss of function in vitro or even in animal models, our findings support that secondary genetic or environmental factors should be carefully assessed in the context of an ultimate human clinical phenotype.

Experimental Procedures

Mice

A knock-in (KI) mouse to mimic the human variant at amino acid 1458 of human ANK2 was generated by The University of Michigan Transgenic Animal Model Core.

A single guide RNA (gRNA) was designed to create a CRISPR-induced indel in a region close to codon 1371 in mouse (which corresponds to 1458 in human sequence, ENSMUST00000182078.9). The gRNA along with Cas9 were microinjected into C57BL/6J and SJL/J zygotes. Chimera mice were screened for the single amino acid substitution at the guide sequence. Germline mice were generated by breeding chimeras to C57BL6/J mice. After germline transmission, mice were genotyped and sequenced to confirm the substitution of the Glutamic acid amino acid (E) for Glycine (G) (Forward: TGTAGACCAGTCCACCAGACACATT; Reverse: CCCTGTCTAATTTCTCTAAAGTCAGAGGC). Mice were backcrossed on a C57BL/6 background for five generations. The whole colony was produced from a single founder. Age-matched male and female mice were used in experiments throughout the manuscript. All animal experiments were conducted in accordance with the Ohio State University Institutional Animal Care and Use Committee guidelines (IACUC Protocol Number 2011A00000034).

Echocardiography

Transthoracic echocardiography using (GE Logiq e) with the L10-22 (mHz) transducer was performed on mice anesthetized using 2% isoflurane in 95% O2 and 5% CO2. Anesthesia was maintained by administration of oxygen and approximately 1.25% isoflurane during the whole imaging procedure. Heart rate (HR) was monitored throughout imaging and recordings taken at a HR less than 400bpm were excluded from the analysis. Mice were immobilized on a heated imaging stage during image acquisition and a temperature probe was inserted into the rectum of the mouse to monitor its core temperature of approximately 37°C. Electrode gel was placed on the ECG sensors of the heated platform. Parasternal long-axis images inclusive of twodimensional loops and freeze-frame end-diastolic images were collected to measure end-diastolic LV cavity dimension (LVID, d), and posterior wall thickness (LVPW, d).

Parasternal short axis images at the level of papillary muscles using M-mode were also collected to determine LVPW and LVID, in both systole (s) and diastole (d), as previously described [START_REF] Cavus | Giant ankyrin-G regulates cardiac function[END_REF]. Parasternal long axis and M mode measurements were used to calculate functional parameters including fractional shortening (FS%) and ejection fraction (EF%).

Electrocardiogram

Surface electrocardiogram analysis was conducted on mice anesthetized with isoflurane (2% in 1L/min oxygen). Anesthesia was maintained by administration of oxygen and approximately 1% isoflurane during the whole recording procedure. Mice were immobilized on a heated imaging stage during acquisition. Lead II ECGs were collected using PowerLab equipment (AD Instruments). Subsurface ECG recordings were collected from anesthetized mice around three months of age for a 5-min interval of recording. Mice at ~three months of age were stimulated with epinephrine (2mg/kg) under isoflurane sedation and ventricular arrhythmic events were calculated over a 30-min period. Conscious ECGs were also collected using implanted radiotelemetry devices using an ETA-F10 miniature telemeter (DSI) and Dataquest A.R.T acquisition software around six months of age for baseline and post stimulation measurements. All conscious ECGs were analyzed using LabChart software.

Researchers blinded to genotype performed data collection and analysis. Conscious mice were stimulated with epinephrine (2mg/kg). After a week of rest, mice were stimulated with epinephrine (2mg/kg) and caffeine (120mg/kg, Millipore Sigma C1778) at the same time. ECG recordings were collected before stimulation to measure baseline ECG features. Mice were then recorded for an hour poststimulation. The total number of ventricular arrhythmic events were calculated by combining the number of arrhythmic events such as bigeminy (>10 consecutive abnormal ventricular beats alternating with normal ventricular beats), ventricular tachycardia (>10 consecutive beats of tachycardia), non-sustained ventricular tachycardia (three to ten consecutive beats), and isolated ventricular arrhythmic events such as premature ventricular contractions (PVCs) and ventricular couplets.

Immunoblotting

Heart tissue samples were isolated from seven-week and six-month old mice after being euthanized using CO2 and homogenized using a Cryolys-cooled Precellys 24 bead homogenizer (Bertin Corp.) using a combination of 1.4mm and 2.8mm ceramic beads at 6000rpm for three bouts of 15 seconds in homogenization buffer (0.025M Tris-HCl, 0.15M NaCl, 0.001M EDTA, 1% v/v NP-40, 5% v/v glycerol, pH=7.4), as previously described [START_REF] Lubbers | Defining new mechanistic roles for alphaII spectrin in cardiac function[END_REF]. Homogenates were then centrifuged for 30 minutes at high speed at 4⁰C. Following quantification by BCA assay (Pierce), 30-40ug of lysates were separated on 4-15% precast ProteanTGX gels (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 hour and then incubated in primary antibody overnight at 4°C. Primary antibodies targeted Ankyrin-B (1:1000, Covance antibody) [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF], Na + /Ca 2+ exchanger 1 (NCX1) (1:1000, Cell Signaling Technology CST 79350), NKA (1:1000, Cell Signaling Technology CST 3010), and Ankyrin-G (1:1000, Covance, a gift from Dr. Paul Jenkins) [START_REF] Jenkins | Giant ankyrin-G: a critical innovation in vertebrate evolution of fast and integrated neuronal signaling[END_REF]. Secondary antibodies used were donkey anti-rabbit or donkey anti-mouse (Jackson ImmunoResearch Laboratories, Bio-Rad). Densitometry analysis was performed using ImageJ software.

Histologic analysis

Adult ventricular myocytes were isolated, and processed for immunofluorescence as previously described [START_REF] El Refaey | Altered Expression of Zonula occludens-1 Affects Cardiac Na(+) Channels and Increases Susceptibility to Ventricular Arrhythmias[END_REF]. For adult ventricular myocytes, staining experiments were performed in solution. Briefly, cells were blocked for 1 hour in blocking solution (3% fish gel, 0.75% triton-100(10%) and 1% DMSO). Cells were incubated in primary antibodies at 4ºC overnight while rotating. Primary antibodies included NCX (Swant R3F1, 1:100), Ankyrin-B (1:100, Covance antibody) [START_REF] Roberts | Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy[END_REF] and NKA (Abcam ab7671, 1:100). Secondary antibodies, including anti-rabbit and antimouse conjugated to Alexa-Fluor, were applied to the samples for 2 hours at room temperature. For control experimental, parallel samples were incubated with secondary antibodies and with primary antibody controls for 2 hours at room temperature and processed to ensure lack of non-specific secondary antibody staining. After secondary antibody incubation for 2 hours, cells were extensively washed, applied to the slides in Vectashield imaging medium (Vector Laboratories, Newark, CA, USA), and then coverslips (#1) were applied. Images were collected on a confocal microscope (LSM 510 Meta, Zeiss, Oberkochen, Germany). Images were collected using identical confocal protocol settings at room temperature, and the observer was blinded to the genotype. Quantification of signal intensity was performed using image J software. Area of overlap between two signals was calculated using ROI (region of interest) manager under Image J software. All calculations were normalized to control.

Detection of cytosolic Ca 2+ by confocal microscopy

Control or AnkBp.E1458G +/+ myocytes were loaded with 10 μM Fluo-4-AM (Thermo Fisher Scientific, MA, United States) for 20 minutes at RT. After 10-20 minutes of de-esterification in free dye solution, the myocytes were mounted in an imaging chamber (Warner Instruments, CT, United States) and continuously superfused with an external solution containing (in mM): 137.0 NaCl, 5.4 KCl, 4.5 MgCl2, 0.16 NaH2PO4, 3 NaHCO3, 20.0 HEPES, 10.0 glucose, 10.4 taurine, 1.8 CaCl2. Intracellular Ca 2+ transients were induced by electrical field stimulation (SD9 stimulator, Grass Technologies/Astro-Med Inc., RI, United States). After stabilization (usually 3-5 min) imaging was performed using Olympus FluoView FV 1000 (Olympus America Inc., PA, United States) confocal microscope system equipped with x60 oil-immersion objective lens (NA 1.4). Fluo-4 was excited with a 488 nm line of argon laser, and the signal was collected at 500-600 nm wavelengths. Linescanning (512 × 512 pixels, 2.0 μS/pixel and 2.1 msec per line) was performed along the longitudinal axis of cells (avoiding nuclei). Fluorescence signals were normalized to the baseline cellular fluorescence (F0). Ca 2+ transients were analyzed as the F/F0 mean value over 10-15 transients for each image. In the experiments with Isoproterenol (ISO) treatment, the drug was administered to the perfusion buffer.

Spontaneous Ca 2+ waves were evaluated during 35 sec after stimulation at 2 Hz. Ca 2+ alternans were induced incrementally by increasing the pacing frequency (1 to 5 Hz).

Transverse aortic constriction

Mice were anesthetized with 2% isoflurane and intubated for artificial ventilation at 120-160 breaths per minute, tidal volume of 0.2-0.35 ml. A Heating pad was used to keep body temperatures at 37 °C throughout the procedure. The procedure was performed on three-month-old mice as previously described [START_REF] Lubbers | Defining new mechanistic roles for alphaII spectrin in cardiac function[END_REF]. Briefly, the transverse aorta was accessed via a left lateral thoracotomy and the aorta was ligated overlying a blunted 27-gauge needle. The needle was removed immediately following ligation leaving a discrete region of stenosis at the aorta. Successful constriction was confirmed by checking the aortic root and the constriction site using echocardiography. The surgeon was blinded to genotype.

Quantitative real-time PCR

Real-time PCR was performed as previously described [START_REF] Cavus | Giant ankyrin-G regulates cardiac function[END_REF]. Briefly, total RNA from the mouse heart tissues was extracted with TRIzol Reagent (Invitrogen) following manufacturer's instructions; 1 μg of total RNA, treated with ezDNase, was used for the first-strand complementary DNA synthesis using SuperScript IV Vilo Master Mix (Thermo Fisher Scientific). qRT-PCR reactions were performed in triplicate in 96-well optical plates with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). Primers for Ank2-F: TACAACCAACGTGTCTGCCA and R: TGCAAAGGCAACAGACTCCT; Nppa-F: 5'-CTGGACTGGGGAGGTCAAC-3', R:

Fast Fourier Transformation (FFT) Analysis

Heart rate variability was measured via fast Fourier transformation (FFT) based time-and frequency-domain analysis. Custom MATLAB script (39) allowed FFT analysis of text files with every raw RR interval within a period of time. To prepare data for MATLAB analysis, ECG recordings were edited on Lab chart, and all noise was removed. Each RR interval was then measured by Lab chart and exported into a text file in 2-minute increments to screen for potential noise missed in the initial edit.

A total of 14 minutes was analyzed for RR and SDRR intervals and also FFT analyzed

Statistics

  Timp1-F: 5'-GGCATCTGGCATCCTCTTGT-3', R: 5'-CAGGTCCGAGTTGCAGAAGG-3'. Hprt levels were used as a normalization control.

  to determine low-frequency (LF) and high-frequency (HF) spectra, LF/HF, etc. (LF range 0.15-1.5 Hz, HF range 1.5-5 Hz).ImmunoprecipitationControl and AnkBp.E1458G +/+ mouse heart samples were homogenized in buffer (containing 0.025 m Tris-HCl, 0.15 m NaCl, 0.001 m EDTA, 1% (v/v) Nonidet P-40, 5% (v/v) glycerol, pH 7.4) using the bead homogenizer. Lysates were centrifuged for 30 min at 13,000 rpm at 4 °C. 400µg of supernatant was incubated and rotated with AnkB-Ig (Covance, custom-made antibody) or control IgG at 4 °C overnight. Following incubation, lysates with antibodies were rotated and incubated with Protein A/G Magnetic Beads (Pierce, number 88802) for 4-5 h at 4 °C. The supernatant was then removed from the beads using a magnetic stand, and the beads were washed 3 times with PBS containing 500mM NaCl. Bound protein was eluted with 2× Laemmli sample buffer and β-mercaptoethanol and heated to 95 °C for 10 min before immunoblotting with NKA (1:750, Cell Signaling Technology CST 3010).

Figure 1 .

 1 Figure 1. Creation of a novel AnkBp.E1458G knock-in mouse model. A.

Figure 8 .

 8 Figure 8. AnkBp.E1458G +/+ mice display severe structural phenotype following

Figure 1

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 9 Figure 9

Figure 10 Click 8

 108 Figure 10

  +/+ mice exhibit electrical phenotypes Conscious six-month old AnkBp.E1458G +/+ mice exhibited sinus bradycardia at rest (mean HR is 658.7±15.2 bpm in control mice vs. 590.5±21.58 bpm in AnkBp.E1458G +/+ mice; Figure

  Data are presented as mean ± SEM. For the comparison of two groups, we performed unpaired two-tailed student t-test when data passed normality test (Shapiro-Wilk normality test). When data did not pass Shapiro-Wilk normality test, we

	performed Mann-Whitney U test (non-parametric for unpaired comparison) to
	compare two groups. For comparison of more than 2 groups, ANOVA test was
	performed when data passed Shapiro-Wilk normality test followed by Tukey's
	multiple comparisons test. When data did not pass Shapiro-Wilk normality test,
	Kruskal-Wallis test (non-parametric) was performed followed by Dunn's multiple
	comparisons test. For cardiac function parameters (EF, FS, LVIDd, LVIDs) measured
	over time in response to TAC, data was screened graphically with model residuals,
	homoskedasticity, and QQ plots to ensure the applicability of a two-way ANOVA. A
	two-way ANOVA mixed effects model for repeated measures was then used with
	post-hoc Holm-Šídák multiple comparisons test. Sphericity was not assumed, and
	the Geisser-Greenhouse correction was deployed. For our study, a value of p<0.05
	was considered statistically significant.

  AnkBp.E1458G +/+ mice show more ventricular arrhythmic events after epinephrine stimulation using implanted telemeters around six months of age (Con, N=15 and AnkBp.E1458G +/+ , N=22). Data did not pass Shapiro-Wilk normality test and non-parametric (Mann-Whitney test) Summary data on Ca 2+ transient amplitude and C. time constant of single exponential decay (Tau) in control and AnkBp.E1458G +/+ myocytes with or without ISO treatment at indicated doses. All groups were compared using paired mixed-effects ANOVA followed by Tukey correction. P<0.05 vs control or AnkBp.E1458G +/+ without Comparison of the fraction of myocytes with spontaneous Ca 2+ waves in 35 seconds post-stimulation at 2 Hz in control and AnkBp.E1458G +/+ mice with and without ISO treatment. The presence or absence of spontaneous Ca 2+ waves was Hz were considered for this analysis. Control and AnkBp.E1458G +/+ in each condition were compared by student's t-test. P<0.05 vs control in the same condition was considered significant. N=4-5 animals per group (n=7-14 myocytes).

	unpaired t-tests were performed (A-G). Data did not pass Shapiro-Wilk normality test
	and non-parametric (Mann-Whitney test) was performed.
	Figure 4. Aged AnkBp.E1458G +/+ mice display heart rate variability at rest. A.
	High frequency (HF) and B. Low frequency (LF) spectral components of heart rate was performed. B. AnkBp.E1458G +/+ mice display significant increase in the number treatment was considered significant. N=5-7 animals per group (n=15-34 myocytes).
	variability HRV. Data did not pass Shapiro-Wilk normality tests and non-parametric of ventricular arrhythmic events after epinephrine and caffeine stimulation using Shapiro-Wilk test was used to evaluate normal distribution. D. Representative
	(Mann-Whitney tests) were performed. C. LF/HF using Fast Fourier transform (FFT) telemeters around six months of age (Con, N=6 and AnkBp.E1458G +/+ , N=6). C. confocal line-scan images of control (upper panel) and AnkBp.E1458G +/+ myocytes
	analysis and radio-implanted telemeters in control and AnkBp.E1458G +/+ mice AnkBp.E1458G +/+ mice exhibit an increase in the number of sustained arrhythmic (lower panel) showing spontaneous Ca 2+ waves in 35 seconds post-stimulation at 2
	around six months of age (Con, N=13 and AnkBp.E1458G +/+ , N=16). Data passed Shapiro-Wilk normality test and unpaired t-test was performed. D. RR-Interval, data passed Shapiro-Wilk normality test and unpaired t-test was performed. E. SDRR-Interval, data did not pass Shapiro-Wilk normality test and non-parametric (Mann-Whitney test) was performed. F. representative traced from control and AnkBp.E1458G +/+ mice over 1 min interval. events after epinephrine and caffeine stimulation (Con, N=6 and AnkBp.E1458G +/+ , N=6). Data passed Shapiro-Wilk normality tests and unpaired t-tests were performed (B-C). D. AnkBp.E1458G +/+ mice display longer episodes of the sustained events after epinephrine and caffeine stimulation (Con, N=6 and AnkBp.E1458G +/+ , N=6). Data did not pass Shapiro-Wilk normality test and non-parametric (Mann-Whitney test) was performed. E. AnkBp.E1458G +/+ mice demonstrate a higher number of non-Hz. E. AnkBp.E1458G +/+ myocytes stimulated at 3 Hz, showing the presence of alternans
	sustained ventricular events (NSVT) after epinephrine and caffeine stimulation (Con, (lower panel). G. Summary data on the fraction of myocytes presenting alternans at
	Figure 5. AnkBp.E1458G +/+ mice display arrhythmias following adrenergic N=6 and AnkBp.E1458G +/+ , N=6). Data did not pass Shapiro-Wilk normality test and 3 Hz or 4 Hz pacing frequency in control and AnkBp.E1458G +/+ myocytes with or
	stimulation/stress. A. AnkBp.E1458G +/+ mice display ventricular arrhythmic events
	non-parametric (Mann-Whitney test) was performed. F-H. Representative ECG without ISO treatment. The presence or absence of alternans was analyzed using a
	after epinephrine stimulation at an early age (~three months of age) under isoflurane
	traces denoting non-sustained and sustained arrhythmic events in the two-tailed Chi-squared test. P<0.05 vs control in the same condition was considered
	sedation (Con, N=6 and AnkBp.E1458G +/+ , N=6). Data did not pass Shapiro-Wilk AnkBp.E1458G +/+ mice after epinephrine and caffeine stimulation. significant. N=4-5 animals per group (n=10-28 myocytes). H. Threshold frequency for
	+/+ mice at normality test and non-parametric (Mann-Whitney test) was performed. B-D.
	seven weeks of age (N=4 mice/genotype), data passed Shapiro-Wilk normality test Representative ECG traces denoting PVCs and chain of couplets in the Figure 7. Calcium handling in AnkBp.E1458G +/+ myocytes. A. Representative the development of alternans. Only myocytes that manifested alternans between 1-5
	and unpaired t-test was performed. E-F. Immunoblotting and quantitative analysis of AnkBp.E1458G +/+ mice at early age after epinephrine stimulation and under confocal line-scan images and Ca 2+ transient profiles recorded in mouse ventricular
	normalized AnkB expression in the control and the AnkBp.E1458G +/+ mice (N=4 isoflurane sedation. myocytes loaded with Fluo-4-AM and stimulated at 1 Hz, with or without 500 nM ISO
	mice/genotype), data passed Shapiro-Wilk normality test and unpaired t-test was

Figure 6. AnkBp.E1458G +/+ mice display a significant pattern of arrhythmia after epinephrine and caffeine stimulation. A. treatment in control (upper panel) and AnkBp.E1458G +/+ (lower panel) myocytes. B. analyzed using a two-tailed Chi-squared test. N=5-7 animals per group (n=12-36 myocytes). F. Representative confocal line-scan images of control (upper panel) and

Acknowledgements

The authors would like to acknowledge the University of Michigan Transgenic Animal Model Core for their help in the generation of the AnkBp.E1458G murine model. The authors would also like to acknowledge the Ohio State University College of Veterinary Medicine Comparative Pathology & Digital Imaging Shared Resource for their assistance with histology services.

Funding

The authors are supported by NIH grants, HL146969 to M.E.R, HL135754 to P.J.M., HL156652 to T.J.H., HL063043, HL074045 to S.G., a grant from the Ohio State Frick Center for Heart Failure and Arrhythmia and the Leducq Foundation (TNE FANTASY 19CV03) to P.J.M. and M.E.M. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Data availability

All data are available within the manuscript or supporting information.

Hearts were excised and fixed in 10% neutral buffered formalin for 24 hours and then stored in 100% ethanol at 4º C until sectioning. Masson's trichrome staining was performed on 5μm sections at the histology core at the Wexner Medical Center at the Ohio State University. Trichrome-stained cardiac sections were collected on an EVOS microscope (Thermo Scientific). Researchers blinded to genotype performed image collection and analysis. Whole-heart images were edited in Adobe Photoshop to excise aortic tissue, as the focus of the study is intra-myocardial fibrosis. Fibrosis was quantified using an add-on to MATLAB (Mathworks). The add-on converts images from the RGB color space into CIELAB color space, then segments images using the kmeans algorithm as previously described [START_REF] Lubbers | Defining new mechanistic roles for alphaII spectrin in cardiac function[END_REF]. Finally, the fibrosis (blue) segment is filtered through a color mask to remove noise, and the ratio of fibrosis to healthy red tissue is calculated.

Isolation of cardiomyocytes

Adult mouse ventricular cardiomyocytes were prepared as previously described [START_REF] Koval | Ca2+/calmodulin-dependent protein kinase II-based regulation of voltage-gated Na+ channel in cardiac disease[END_REF]. Murine hearts from male and female mice were obtained after animals were euthanized by CO2 asphyxiation followed by cervical dislocation in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and the Ohio State University Institutional Animal Care and Use Committee-approved protocols. Following isolation, cells were fixed in ethanol and stored at -20ºC till staining experiments.

Immunofluorescence

Supporting information

This article contains supporting information.

Conflict of Interest

The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions

M.E.R. and P.J.M. conceived and designed the project; M.E.R., M.J.W., J.