Iron overload decreases CaV1.3-dependent L-type Ca2+ currents leading to bradycardia, altered electrical conduction, and atrial fibrillation

Abstract

Background: Chronic iron overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy characterized by contractile dysfunction and electrical disturbances, including slow heart rate (bradycardia) and heart block.

Methods and results: We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca(2+) current (I(Ca,L)) densities and positive (rightward) voltage shifts in I(Ca,L) activation. Pacemaker current (I(f)) was not affected by CIO. Because I(Ca,L) in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of Ca(V)1.3 channels (in addition to Ca(V)1.2 channels), our data suggest that elevated iron preferentially suppresses Ca(V)1.3 channel function. Consistent with this suggestion, CIO reduced Ca(V)1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in Ca(V)1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in Ca(V)1.3 knockout mice.

Conclusions: Our results demonstrate that CIO selectively reduces Ca(V)1.3-mediated I(Ca,L), leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.

Figure 1

Effects of chronic iron-overload on HR in conscious mice. A, Representative telemetry ECG recordings (1 second duration) before iron injection (i.e. baseline) and after 2 and 4 weeks of injection with iron or placebo. Sample ECGs were measured at 2 PM (during mouse sleeping) to control for circadian rhythms. B, HR measured every 4 hours over a consecutive 48 h period illustrating prominent circadian rhythms. C, Summary HR data averaged over a 4 week period in control conditions and following iron-overload. For panels B and C data are means±SEM; n = 5–7 mice per group. *P<0.05 compared to control at the same time point analyzed by Student’s t-test.

Figure 2

Effects of chronic iron-overload on ECGs measured in anesthetized mice following autonomic nervous system blockade by intraperitoneal injection of atropine (1 mg/kg) and propranolol (10 mg/kg) in isolated Langendorff-perfused hearts. A, Representative ECGs (1 second recordings) in control conditions and in iron-overload at baseline (pre-injection) as well as 25 min after injection of atropine and propranolol. B, Summary data showing HR at baseline and following autonomic nervous system blockade. Data are means±SEM, n=7 mice in each group. HR was reduced (P<0.05) at all time points in iron-overload mice compared to controls (Student’s t-test). C, Summary data showing averaged HRs in isolated Langendorff-perfused hearts from control and iron-overload mice. Data are means±SEM, n=10–11 hearts per group, *P<0.05 vs. control, by Student’s t-test.

Figure 3

A, representative spontaneous action potentials recorded from isolated SAN myocytes in control condition and following iron-overload. B, Summary data illustrating the effects of iron loading on spontaneous AP frequency, diastolic depolarization slope and APD₉₀. Data are means±SEM, n=10 myocytes in each group, *P<0.05 vs. control by Student’s t-test. See also Supplemental Table SIII.

Figure 4

Properties of the hyperpolarization-activated current, I_f, in SAN myocytes from CIO mice. A, Representative I_f recordings in SAN myocytes. Voltage-clamp protocol is shown in the inset. B, Summary I_f I-V curves showing no significant difference between control and iron-overload conditions. Data are means±SEM, n=5 control myocytes and n=6 iron-overload myocytes.

Figure 5

Effects of CIO on I_Ca,L in SAN and right atrial myocytes. Ca_V1.2 and Ca_V1.3-dependent I_Ca,L was measured simultaneously (see Methods and inset in panel A). A, Representative I_Ca,L recordings in SAN myocytes. B, I_Ca,L I-V curves in sinoatrial node myocytes. Data are means±SEM, n=8 myocytes for each group; P<0.05 compared to control at the same membrane potential analyzed by Student’s t-test. C, I_Ca,L activation curves in SAN myocytes. D, Representative I_Ca,L recordings in working right atrial myocytes. E, I_Ca,L I-V curves in working right atrial myocytes. Data are means±SEM, n=7 atrial myocytes for control and n=11 atrial myocytes for iron-overload; *P<0.05 compared to control at the same membrane potential analyzed by Student’s t-test. F, I_Ca,L activation curves in working right atrial myocytes.

Figure 6

A, Real-time PCR measurements of mRNA level for the Ca_V1.2 and Ca_V1.3 alpha subunits of the L-type Ca²⁺ channels. The results show the ratio of mRNA in iron-loaded atria versus placebo control for Ca_V1.2 and Ca_V1.3. n=5 for all groups. *P<0.05 compared to 1 using the Student’s t-test. B, Representative Western Blot for Ca_V1.2 and Na⁺/K⁺ ATPase (loading control) in atria isolated (top) along with the relative protein expression levels of Ca_V1.2 normalized by the level of Na⁺ pump expression for control and iron-overload atria. n=3 for control and iron-overload. #P<0.05 relative to control, Student’s t-test.

Figure 7

Effects of iron treatment in conscious Ca_V1.3 knockout mice. A, Representative telemetry ECGs (0.5 second recordings) before (baseline) and after (3 weeks) iron injection in Cav1.3 knockout and littermate wild-type mice. All sample ECGs were measured at 2 PM (sleeptime). B, HRs averaged over a 48 hour period (measured every 4 hours) for Cav1.3 knockout and littermate wild-type mice showing increases in HR for Cav1.3 knockout mice but decreases in wild-type littermate controls. C, Summary of PR-intervals after 3 weeks of iron injection in Cav1.3 knockout and littermate wild-type mice. For panels B and C data are means±SEM; n>7 mice per group. +P<0.001 compared to baseline (time = 0) analyzed by Kruskal-Wallis test with Dunn’s posthoc analysis. *P<0.05 compared to wild-type littermates at the same time point using Student’s t-test.

Figure 8

A. Representative ECG recordings illustrating conduction disturbances observed in anesthetized CIO mice after 3 weeks of iron injection: top trace sinus pause, middle PR interval prolongation (1^st degree ANV block) and bottom trace 2:1 AVN block. No conduction disturbances were observed in control mice. B. Average PR-intervals at baseline (before iron injection) and after 3 weeks of iron injection in anesthetized mice and hearts excised from iron-overload mice.