The experimental procedures in mice and the protocol used in this study were approved by the University of Illinois at Chicago (UIC) Animal Care Committee (ACC No. 19-178). All animal studies were performed according to approved guidelines for the use and care of live animals, and conformed with animal care policies and procedures of UIC. The HVCN1^−/− and wild-type (C57BL/6 background) mice were used in the study. Mice were housed in temperature- and humidity-controlled rooms with 12-h light-dark cycles in the animal care facility at the UIC. We studied male mice, similarly aged HVCN1^−/− mice (n = 4, 3 months old) and wild-type control mice (n = 4, 3 months old) were euthanized and heart samples were isolated for analysis.

The RNA-seq analysis was performed as previously described (Hong et al., 2021). We first extracted RNA with Maxwell^® RSC simplyRNA Cells Kit (Promega AS1390) based on manufacturer’s instructions, using Maxwell^® RSC Instrument (Promega AS4500), and then RNA was quantified using Qubit 4.0 Fluorometer (Invitrogen) with the Qubit RNA HS Assay Kit (REF Q32855) analyzed for integrity using the Agilent 4200 TapeStation RNA ScreenTape assay (Agilent 5067–5576, 5067–5577). RNA samples were normalized to 250ng, and library prep was carried out using the universal Plus mRNA-Seq kit (NuGen 0520-A01) as written in the product manual (NuGen M01485 v5). In brief, RNA underwent poly-A selection, enzymatic fragmentation, and generation of double-stranded cDNA using a mixture of Oligo (dT) and random priming. The cDNA underwent end repair, ligation of dual-index adaptors, strand selection, and 15 cycles of PCR amplification. We determined the number of cycles by qPCR of a small aliquot of un-amplified libraries. All intermediate purification steps, and final library purification were carried out using Agencourt AMPure XP Beads (Beckman Coulter A63881). We measured purified library concentrations with the Qubit 1X dsDNA HS Assay Kit (Invitrogen Q33231), and fragment size distribution was confirmed using the D5000 ScreenTape assay (Agilent 5067–5588, 5067–5589), and libraries were pooled in equimolar amounts based on the Qubit concentration and TapeStation average size and run on MiniSeq for index balancing. The libraries were re-pooled with corrected inputs based on the % Reads Identified (PF) results from the MiniSeq run, and the new pool was purified with the Agencourt AMPure XP Beads (Beckman Coulter A63881). The final, purified pool was quantified by qPCR using the KAPA Library Quantification Kit and run on a NovaSeq6000 SP flow cell, 2 × 50 nt, one lane, at the University of Illinois Roy J. Carver Biotechnology Center High-Throughput Sequencing and Genotyping Unit. Raw reads were aligned to the reference genome hg38 using STAR (Dobin et al., 2013). ENSEMBL gene expression was quantified using FeatureCounts (Liao et al., 2014). Normalized and differential expression statistics were computed using edgeR (Robinson et al., 2010; McCarthy et al., 2012), and p-values were adjusted for multiple testing using the false discovery rate (FDR) correction of Benjamini and Hochberg. We performed unsupervised hierarchical clustering of all differentially expressed genes using the Euclidean distance and complete linkage method, and generated volcano plots using R. Up- and down-regulated genes were analyzed separately using the DAVID functional annotation tool and the Gene Ontology Biological Process (GO BP) database. Bar charts of the top 20 most significant upregulated and downregulated pathways, based on FDR-corrected p-value, were constructed with the ggplot2 R package. We constructed heatmaps for the cardiac conduction and calcium ion transport into cytosol pathways with the ComplexHeatmap R package.

RT-PCR analysis was performed as previously described (Kim et al., 2014). Total RNA was isolated from hearts of adult wild-type or HVCN1^−/− mice using Trizol RNA extraction agent (Invitrogen) according to the manufacturer’s instructions. For the reverse transcription, total RNA was quantified using the Nanodrop spectrophotometer (Thermofisher Scientific). Synthesis of cDNA was carried out with SuperScript IV RNase Reverse transcriptase (Invitrogen) and primers with 2 μg of total RNA as template. Real-time PCR was performed on an Applied Biosystems (Thermofisher Scientific) to determine mRNA levels of differentially expressed genes according to the manufacturer’s recommended protocol using SYBR green assays. For each group, data were collected from three independent samples; three replicas were performed for each sample. The primer sequences used for SYBR green-based fluorescence were GAPDH: 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′ and 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′; NOX1: 5′-TTC​CTC​ACT​GGC​TGG​GAT​AG-3′ and 5′-AGT​CCG​AGG​GCC​ACA​TAA​GA-3′; NOX2: 5′-TGG​CGA​TCT​CAG​CAA​AAG​GTG​G-3′ and 5′-GTA​CTG​TCC​CAC​CTC​CAT​CTT​G-3′; NOX4: 5′-TCT​GGA​AAA​CCT​TCC​TGC​TG-3′ and 5′-CCG​GCA​CAT​AGG​TAA​AAG​GA-3′; SLC4A1: 5′-CCG​TGA​ACT​CTT​CAT​TGC​TGC​C-3′ and 5′-ACC​AGG​AAC​AGC​AAG​CTC​ATG​C-3′; SLC4A2: 5′-GCA​CCT​CCA​TTC​TGT​TTG​CGG​T-3′ and 5′-GCT​CAC​GAA​CTT​CCA​ACA​CAG​C-3′; SLC4A3: 5′-GGT​GCT​GAT​TGC​CTT​CTC​CAG​T-3′ and 5′-GAC​AAC​GAA​GCC​AGA​GGA​GAA​G-3′; SLC26A6: 5′-TAC​CGT​GTG​GAC​AGT​AAC​CAG​G-3′ and 5′-CCT​GTA​CCA​AGC​TCC​GAG​ACA​T-3′.

All data were presented as the mean ± SEM. The normality in each group was determined by the Shapiro-Wilk test, and p > 0.05 was considered to indicate normally distributed. Homoscedasticity was determined by the two-sample F test. For equal variances between two groups, significance between means was determined by Student’s t-test. For unequal variances between groups, significance between means was determined by Welch’s t-test. p < 0.05 was considered to indicate a statistically significant difference.

To explore the potential function of HVCN1 in the heart, we performed a transcriptomic analysis of wild-type (WT) and HVCN1^−/− mouse hearts. The HVCN1 null hearts exhibited a significantly different transcriptome profile compared with WT hearts (Figure 1 and Supplementary Table S1). Hierarchical clustering of differentially expressed genes (DEGs) in WT and HVCN1^−/− hearts showed two main clusters with samples of the same group clustered together (Figures 1A,B). A total of 206 DEGs were identified with 67 downregulated and 139 upregulated (Figure 1C). The major Gene Ontology (GO) categories identified by RNA sequencing and the most significantly modulated in the HVCN1^−/− mouse hearts included immunological activity, ion and receptor binding, metabolic process, and ion channel activity (Figures 1D,E).

It is noted that both oxidoreductase activity and oxidation-reduction process were downregulated in HVCN1 null hearts from the GO enrichment analysis of top significant gene groups (Figure 1D). The oxidoreductase utilizes NADP⁺ or NAD⁺ as cofactors to catalyze the transfer of electron between molecules, and HVCN1 provides charge and pH compensation during the NADPH oxidoreductases (NOXs) activation (Seredenina et al., 2015). Since HVCN1 was coupled with NOX activity, we determined the effects of HVCN1 deletion on the expression of NOX family. We detected three isoforms of the NOX family in the hearts, including NOX1, NOX2, and NOX4 (Figure 2). The RNA-seq results showed that three isoforms were downregulated in HVCN1 null hearts (Figures 2A–C). RT-PCR confirmed changes in RNA-seq, and the mRNA expression levels of all three NOX isoforms were markedly reduced in the HVCN1^−/− hearts compared with the ones in the WT hearts (Figures 2D–F), indicating that deletion of HVCN1 was associated with gene expression of the NOX family.

The HVCN1 has been proposed to combine with HCO₃ ⁻ exchangers in regulating transport-mediated CO₂ disposal in the heart (Vairamani et al., 2017). We assessed the expression of bicarbonate transporters in the HVCN1 null hearts. The SLC4A1 (encoding Anion Exchanger 1, AE1), SLC4A2 (encoding Anion Exchanger 2, AE2), SLC4A3 (encoding Anion Exchanger 3, AE3), and SLC26A6 are the most abundant Cl⁻/HCO₃ ⁻ exchangers expressed in hearts (Figures 3A–D). The RNA-profiling and RT-PCR results showed HVCN1^−/− significantly downregulated SLC26A6 (Figures 3D–H), but did not significantly affect the AE1 and AE3 (Figures 3A,C,E, G). Although RNA-seq data indicated increased AE2 in HVCN1 null hearts (Figure 3B), the RT-PCR analysis showed no significant alterations of AE2 expression between groups (Figure 3F). These data suggested that HVCN1 might affect the expression of SLC26A6 in the regulation of cardiac CO₂ homeostasis.

The effects of HVCN1 proton channel deletion on other proton channels/exchangers and proton-coupled transporters/antiporters were investigated. The sodium-hydrogen exchanger 1 (NHE1) has been reported to regulate intracellular pH in cardiomyocytes (Sundset et al., 2003). Our RNA-seq revealed that NHE1 was upregulated in HVCN1 null hearts. The increased expression of NHE1 might be a functional compensation for the H⁺ transport burden due to the deletion of the HVCN1 proton channel (Figure 4A). Additionally, proton pump (V-type proton ATPase, ATP6V0A1) and proton channel otopetrin 1 (OTOP1) were not altered in the HVCN1 null hearts (Figures 4B,C).

We further assessed the expression of acid-sensing ion channels (ASIC1, ASIC2, ASIC3, ASIC4), proton activated chloride channel (TMEM206), proton-coupled metal-ion transporters (SLC11A1, SLC11A2), proton-coupled amino acid transporters (SLC36A1, SLC36A2, SLC36A4), and proton/amine antiporters (SLC18A1, SLC18A2). Except for ASIC3, there were no notable changes in those genes associated with the HVCN1 deletion (Figures 5A–L). Moreover, the proton-dependent oligopeptide transporters showed differential expression linked with the HVCN1^−/−. Although the expression of SLC15A2 was not changed, the expression of SLC15A3 was decreased, and SLC15A4 was increased (Figures 5M–O), indicating a role of HVCN1 in differential regulation of proton-dependent oligopeptide transport.

We evaluated the effects of HVCN1 deletion on the expression profiles of cardiac ion channels, including cardiac sodium channel alpha subunit (SCN5A), sodium channel beta subunits SCN1B and SCN2B, L-type and T-type calcium channels (CACNA1C, CNCNA1G), I_Ks, I_Kr, I_to, I_K1, I_Kur potassium channels (KCNQ1, KCNH2, KCNE1, KCNE3, KCNE4, KCND2, KCND3, KCNA4, KCNJ2, KCNJ12, KCNA5), I_f channels (HCN2, HCN4), and sarcoplasmic reticulum calcium handling proteins (RYR2, SERCA pump) (Figure 6). The knockout hearts exhibited upregulation of L-type calcium channel CACNA1C, potassium channel beta subunit KCNE3, I_Kur potassium channel KCNA5, I_f channel HCN2 (Figures 6D,I, P, Q), and downregulation of SERCA pump ATPA2 (Figure 6T). The data supported that HVCN1 was involved in the regulation of cardiac electrophysiology, and HVCN1 deletion potentially remodeled action potential profiles in cardiomyocytes.