TRPM7 current in cultured murine embryonic ventricular myocytes. (A) Representative TRPM7 current measured in cultured EVM compared with AVM after full run-up. (B) Mean TRPM7 current density at +100 mV in AVM (I_{Trpm7, AVM} = 8.3 ± 0.9 pA/pF, n = 5) compared with EVM (I_{Trpm7, EVM} = 64.2 ± 16.7 pA/pF, n = 5). (C) Interference contrast (DIC, Left) and fluorescence GFP image (Right). (D) PCR across exon 17 from genomic DNA isolated from Trpm7^fl/fl EVM transduced with Ad-TnT-Cre (1), Ad-CMV-Cre (2), and Ad-CMV-Lacz (3), Trpm7^fl/fl fibroblasts (+), and Trpm7^fl/- tail (-). Black arrow, full-length exon 17. Red arrow, deleted exon 17. (E) Representative TRPM7 current measured in Trpm7^fl/fl EVM and Trpm7^fl/fl EVM 5 d after transduction with Ad-CMV-Cre. (F) Mean TRPM7 current density in Trpm7^fl/fl EVM treated with Ad-CMV-Cre (n = 3) compared with untreated Trpm7^fl/fl EVM (n = 5). *P < 0.05.

Trpm7 deletion in embryonic myocardium disrupts cardiac automaticity in vitro. (A) Ca²⁺ transients measured by high-speed line-scanning confocal microscopy in Trpm7^fl/fl EVM 4–5 d after transduction with Ad-CMV-Lacz (Top), Ad-CMV-Cre (Middle), and Ad-TnT-Cre (Bottom). (B) Mean Ca²⁺ transient frequency, (C) cycle length, C_L, (D) peak Ca²⁺ transients, F/F_o, and (E) Ca²⁺ transient length in Ad-CMV-Lacz, Ad-CMV-Cre, and Ad-TnT-Cre treated Trpm7^fl/fl EVMs. *P < 0.05, **P < 0.01.

Trpm7 deletion in vivo disrupts automaticity in zebrafish and induces SPs and AVB in mice. (A) (Left) Images of zebrafish embryos: water-injected (Upper) and Trpm7 MO-injected (Lower). (Right) Trpm7 MO zebrafish (n = 30) and water-injected zebrafish (n = 27) heart rates. (B) Normal sinus rhythm (p denotes P waves; atrial depolarization) with intact atrioventricular conduction in the ECG of a telemetered, conscious WT mouse. (C) Representative ECG showing an episode of SP observed in a KO^αMHC-Cre mouse. Solid arrows denote location of expected p waves. (D) Representative ECGs demonstrating AVB observed in KO^αMHC-Cre mice (broken black arrow, conducted QRS complexes; broken gray arrow, expected location of QRS complex). (E and F) Box plots with overlying data points showing the distribution of the frequency of (E) SPs and (F) AVB observed over 24 h of telemetric monitoring in WT (n = 7) and KO^αMHC-Cre (n = 8) mice. (G) Mean heart rates of WT and KO^αMHC-Cre over a 24-h period were not statistically different. In box plots, error bars represent the SD of the mean. Box height represents the SE. **P < 0.01, ***P < 0.001.

TRPM7 is highly expressed in murine SAN. (A) Confocal images of an isolated SAN freshly dissociated from adult αMHC-Cre-ROSA26^mTmG heart: differential interference contrast (Left) and fluorescence GFP image (Right). (Scale bar, 50 μm.) (B) PCR across exon 17 from genomic DNA isolated from dissected KO^αMHC-Cre and WT SAN. Tail DNA from Trpm7^fl/- (-) serves as a positive control for Trpm7 exon 17 deletion. Black arrow, full-length exon 17. Red arrow, deleted exon 17. (C) Representative TRPM7 current-voltage traces measured in WT SAN, WT AVM, and KO^αMHC-Cre SAN. (D) Mean TRPM7 current density in WT SAN (I_{Trpm7, WT SAN} = 32.0 ± 6.2 pA/pF, n = 6), WT AVM (I_{Trpm7, AVM} = 8.3 ± 0.9 pA/pF, n = 5), and SAN (I_{Trpm7, KOαMHC-Cre SAN} = 0.4 ± 0.2 pA/pF, n = 4, P < 0.01).

Trpm7 deleted SAN exhibit impaired automaticity with slowed diastolic Ca²⁺ rise. Ca²⁺ transients measured by high-speed line-scanning confocal microscopy in (A) WT SAN and (B) KO^αMHC-Cre SAN under basal conditions (Top) and after stimulation with ISO: 2 nM (Middle) and 100 nM (Bottom). (C) Mean Ca²⁺ transient frequency and (D) Ca²⁺ transient length in WT SAN compared with KO^αMHC-Cre SAN under basal conditions and after stimulation with 2 nM and 100 nM ISO. (E) Representative Ca²⁺ transients from WT (Left) and KO^αMHC-Cre (Right) showing the slope of diastolic Ca²⁺ rise (Ca²⁺ ramp). (F) Mean slope of the diastolic Ca²⁺ ramp in WT and KO^αMHC-Cre under increasing ISO concentrations. *P < 0.05, **P < 0.01.

Postnatal SAN/AVN restricted Trpm7 deletion recapitulates the phenotype of global cardiac Trpm7 KO. (A) SAN-restricted Trpm7 deletion (KO^Hcn4-CreERT2) was achieved in Hcn4-CreERT2 × Trpm7^fl/fl/Trpm7^fl/- mice at 6–8 wk of age by treating with tamoxifen (40 mg/kg) by gavage for 4 d, followed by >2-wk washout period before telemetry and SAN studies (B) (Upper) Transmitted light + GFP fluorescence image of Hcn4-CreERT2-ROSA26^mTmG right atrium after tamoxifen treatment. (Lower) Fluorescence mTomato/mGFP image showing recombination (mGFP) restricted to SAN region. (Scale bar, 1 mm.) (C) PCR across exon 17 from genomic DNA isolated from dissected KO^Hcn4-CreERT2 and WT SAN. Tail DNA from Trpm7^fl/- serves as a positive control for Trpm7 exon 17 deletion (-). (D) Representative TRPM7 current–voltage traces measured in WT SAN and KO^Hcn4-CreERT2 SAN. (E) Mean TRPM7 current density in WT SAN and KO^Hcn4-CreERT2 SAN. (F) Representative ECG showing an episode of SP observed in a telemetered KO^Hcn4-CreERT2 mouse. Solid arrows denote location of expected p waves. Red dashed circles and Insets above show change in p-wave morphology after SP, indicative of ectopic atrial focus. (G) Box plots with overlying data points showing the distribution of the frequency SPs (Left) and AVB (Right) observed over 24 h of telemetric monitoring in WT (n = 7) and KO^Hcn4-CreERT2 (n = 5) mice. (H) I_f current–voltage relationship from WT SAN (n = 11) compared with KO^Hcn4-CreERT2 SAN (n = 25). (I) Box plots with overlying data points showing the mean and distribution of I_f current densities at 160 mV from WT SAN (n = 11) compared with KO^Hcn4-CreERT2 SAN (n = 25). *P < 0.05, **P < 0.01.