Complete heart block and sudden death in mice overexpressing calreticulin

Abstract

The expression of calreticulin, a Ca(2+)-binding chaperone of the endoplasmic reticulum, is elevated in the embryonic heart, and because of impaired cardiac development, knockout of the Calreticulin gene is lethal during embryogenesis. The elevated expression is downregulated after birth. Here we have investigated the physiological consequences of continued high expression of calreticulin in the postnatal heart, by producing transgenic mice that overexpress the protein in the heart. These transgenic animals exhibit decreased systolic function and inward I(Ca,L), low levels of connexin43 and connexin40, sinus bradycardia, and prolonged atrioventricular (AV) node conduction followed by complete heart block and sudden death. We conclude that postnatal downregulation of calreticulin is essential in the development of the cardiac conductive system, in particular in the sinus and AV nodes, when an inward Ca(2+) current is required for activation. This work identifies a novel pathway of events, leading to complete heart block and sudden cardiac death, which involves high expression of calreticulin in the heart.

Figure 1

Generation of cardiac-specific calreticulin overexpresser transgenic mice. (a) Calreticulin cDNA encoding full-length protein including the NH₂-terminal signal sequence and the COOH-terminal KDEL ER retrieval signal was subcloned downstream from α-cardiac MHC promoter. The arrows indicate the PCR primers used for recognition of the transgene. (b–d) Western blot analysis of proteins extracted from control and transgenic mice. The blot was probed with the anti-HA antibodies (b and c) or with the rabbit anti-calreticulin antibodies (d). (e) Kaplan-Meier survival analysis of calreticulin transgenic mouse lines. hGH, human growth factor polyA sequences.

Figure 2

Pathological changes in calreticulin transgenic mice. (a and b) Gross morphology of control and transgenic 7-day-old mouse hearts, respectively. No significant morphological changes are observed. (c–f) Gross morphology of transgenic and control 14-day-old mouse hearts (line M148). (c and d) Transverse sections. (e and f) Sagittal sections. (g and h) High magnification images of control and transgenic 14-day-old mouse hearts stained with H&E. (i–l) Sections of transgenic and control (wild-type) cardiac tissue were immunostained with anti-calreticulin (anti-CRT) (i and j) or anti-calsequestrin (anti-CSQ) (k and l) antibodies.

Figure 3

Northern and Western blot analysis of transgenic hearts. (a) Northern blot analysis of mRNA isolated from transgenic and control ventricle of 11-day-old calreticulin transgenic mice. The following cDNA probes were used: ANP, BNP, α-MHC, and GAPDH. (b) Western blot analysis of expression ER-resident chaperones and SR proteins in transgenic hearts. CNX, calnexin; BiP, immunoglobulin binding chaperone; PDI, protein disulfide isomerase; CSQ, calsequestrin. (c) Western blot analysis of expression of phospholamban, α skeletal actin, and β-MHC in transgenic and control mice. (d) Northern blot analysis of mRNA isolated from transgenic and control ventricle of 17-day-old transgenic mice. The Cx43, Cx40, and GAPDH cDNA probes were used. (e) Western blot analysis of expression of Cx43 in transgenic and control hearts. Cx43 and Cx43-P indicate the location of connexin43 and phosphorylated connexin43, respectively. The location of Bio-Rad Laboratories molecular markers is indicated.

Figure 4

M-mode echoes from 17-day-old transgenic and control mice. LV, left ventricle.

Figure 5

Electrocardiogram from calreticulin transgenic mice. (a) Representative traces of electrocardiogram from transgenic and control mice of different ages. (b) Heart rates of nonsedated transgenic (M148) and control mice. The number of measurements is indicated inside the bar. ^AP < 0.001. (d) Progressive AV node disease: from 11-day-old heart (upper recording) to 18-day-old heart (middle recording) to sudden death (lower recording) (20-day-old heart). P, P wave indicating atrial activation; QRS, QRS complex indicating ventricular activation; P-R interval, time of onset of P wave to onset of QRS interval.

Figure 6

Patch clamp for L-type Ca²⁺ channel. The density of I_Ca,L in the ventricular myocytes in control and transgenic mice. The representative current trace families of I_Ca,L in control (a) and transgenic (b) mice. (c) I-V relationships of peak in control (filled circles) and transgenic (open circles) mice. Data are presented as mean ± SEM. ^AP < 0.05.