Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy

  1. Prince J. Kannankeril*,†,
  2. Brett M. Mitchell†,‡,
  3. Sanjeewa A. Goonasekera†,§,
  4. Mihail G. Chelu‡,
  5. Wei Zhang,
  6. Subeena Sood‡,
  7. Debra L. Kearney‖,
  8. Cristina I. Danila‡,
  9. Mariella De Biasi**,
  10. Xander H. T. Wehrens‡,††,
  11. Robia G. Pautler‡,
  12. Dan M. Roden,
  13. George E. Taffet††,
  14. Robert T. Dirksen§,
  15. Mark E. Anderson‡‡, and
  16. Susan L. Hamilton‡,§§
  1. Departments of *Pediatrics and
  2. Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232;
  3. Departments of ‡Molecular Physiology and Biophysics,
  4. ‖Pathology,
  5. **Neuroscience, and
  6. ††Medicine, Baylor College of Medicine, Houston, TX 77030;
  7. §Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14627; and
  8. ‡‡Departments of Medicine and Physiology and Biophysics, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242

  1. Edited by Andrew R. Marks, Columbia University College of Physicians and Surgeons, New York, NY, and approved June 12, 2006

  2. ↵ †P.J.K., B.M.M., and S.A.G. contributed equally to this work. (received for review January 11, 2006)

 
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Abstract

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2 R176Q/+ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2 R176Q/+ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2 R176Q/+, but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2 R176Q/+ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2 R176Q/+ mice, but not in controls. Isolated cardiomyocytes from RyR2 R176Q/+ mice exhibited a higher incidence of spontaneous Ca2+ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

The cardiac ryanodine receptor 2 (RyR2) regulates calcium release from the sarcoplasmic reticulum in cardiomyocytes (1). Two inherited arrhythmogenic syndromes have been linked to mutations in RyR2, arrhythmogenic right ventricular dysplasia (ARVD) and catecholaminergic polymorphic ventricular tachycardia (CPVT) (2, 3). ARVD and CPVT are both characterized by ventricular arrhythmias and a high rate of juvenile sudden death. Patients with CPVT exhibit catecholamine-induced bidirectional ventricular tachycardia (VT) in the setting of a structurally normal heart, whereas patients with ARVD exhibit progressive fibrofatty replacement of the right ventricular myocardium in addition to polymorphic VT. ARVD arising from RyR2 mutations (ARVD2) is typically associated with exercise-induced ventricular arrhythmias and relatively mild structural abnormalities compared with other forms of ARVD and, in some ways, mimics the CPVT phenotype. In fact, the diagnosis of ARVD2 in patients with RyR2 mutations is controversial because of the differences in degree of cardiac structural abnormalities between ARVD2 and other forms of ARVD (4).

Disease-causing mutations in RyR2 and the skeletal muscle isoform RyR1 cluster in three highly conserved regions: a cytosolic N-terminal region, a cytosolic central region, and a C-terminal portion containing the transmembrane and pore regions of the channel (5, 6). Multiple mutations in RyR2 have been reported in patients with ARVD2 (7). Families with the RyR2 R176Q mutation also harbor a second RyR2 mutation, T2504M. The functional consequences of these mutations, both individually and in combination, have been studied in vitro and reveal a “gain of function” represented by an increased probability of channel opening (8–10). Despite similar in vitro effects, the respective contribution of each individual mutation to the overall phenotype is unknown.

The R176Q mutation in RyR2 corresponds to the R163C mutation in RyR1, which causes malignant hyperthermia and central core disease, suggesting that the R176Q mutation alone may be sufficient to alter RyR function and elicit a cardiac phenotype. We developed mice with the R176Q RyR2 mutation to investigate the role of this mutation in the development of cardiomyopathy and arrhythmic susceptibility to catecholamines. By using homologous recombination in ES cells, the R176Q point mutation in RyR2 was engineered in mice by using a knockin strategy (Fig. 1). Because ARVD and CPVT are autosomal dominant disorders, heterozygous (RyR2 R176Q/+) mice were studied. RyR2 R176Q/+ mice exhibited reduced right ventricular end-diastolic volumes but lacked structural abnormalities of the right ventricle. RyR2 R176Q/+ mice also demonstrated VT after injection of caffeine and epinephrine and with programmed ventricular stimulation combined with the β-adrenergic receptor agonist isoproterenol. Additionally, individual cardiomyocytes from RyR2 R176Q/+ mice showed an increased incidence of spontaneous Ca2+ oscillations. These findings identify the R176Q RyR2 mutation as sufficient to cause cardiac dysfunction and catecholamine-dependent arrhythmias and support an overlap between ARVD2 and CPVT due to mutations in RyR2.

Fig. 1.
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Fig. 1.

Targeted engineering of R716Q mutation in the mouse RyR2 locus. (A) The R176Q mutation along with a new RsrII site was introduced in the RyR2 gene. (pGK-Neo/Tet and pCM-TK cassettes were used as positive and negative selection markers, respectively. Lox P sites are indicated as black triangles.) (B) Allele-specific PCR analysis and restriction digestion. A 765-bp fragment was amplified by PCR, and the product was digested with RsrII. WT band, 765 bp; mutant band, 410 bp and 355 bp. (C) Sequencing of an amplified DNA fragment from a correctly targeted ES cell clone shows the presence of the R176Q mutation and the new RsrII restriction site on one allele.


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Results

Function, but Not Structure, Is Altered in RyR2R176Q/+ Mouse Hearts.

Histologic studies revealed no indications of fibrofatty infiltration or fibrosis in hearts from young (16–22 weeks of age) or old (51–57 weeks of age) RyR2 R176Q/+ or WT mice (Fig. 7, which is published as supporting information on the PNAS web site; n = 3–7 for each group). Statistically, there were no differences in left or right ventricular volumes of histological slices from RyR2 R176Q/+ or WT mice as measured by planimetry (data not shown). There were no differences in heart rate under isoflurane during the Doppler ultrasound studies: RyR2 R176Q/+, 359 ± 113 beats per min vs. WT, 383 ± 54 beats per min; P > 0.05. However, systolic function was modestly reduced in RyR2 R176Q/+ mice, as demonstrated by decreased peak aortic velocity: RyR2 R176Q/+, 92 ± 15 cm/s vs. WT, 114 ± 15 cm/s; P < 0.05 and mean aortic velocity: RyR2 R176Q/+, 21 ± 5 cm/s vs. WT, 28 ± 5 cm/s, P < 0.05 (Fig. 8, which is published as supporting information on the PNAS web site).

Using MRI, we found no differences in left or right ventricular end-systolic volumes in 8-week-old RyR2 R176Q/+ mice compared with WT (Fig. 2). However, right ventricular end-diastolic volume was decreased significantly in RyR2 R176Q/+ mice: RyR2 R176Q/+, 25.5 ± 1.6 mm3 vs. WT, 31.9 ± 1.3 mm3; P < 0.05. There was a trend toward lower left ventricular end-diastolic volumes in RyR2 R176Q/+ mice compared with controls, although this trend did not reach significance: RyR2 R176Q/+, 41.8 ± 2.2 mm3 vs. WT, 47.9 ± 1.7 mm3; P = 0.056.

Fig. 2.
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Fig. 2.

MRI reveals decreased right ventricular end-diastolic volume in RyR2 R176Q/+ mice. (A) Representative end-diastolic MRI images for two WT and two littermate RyR2 R176Q/+ mice. (B) Right ventricular end-diastolic volume was decreased in RyR2 R176Q/+ mice (n = 6) compared with WT mice (n = 6). Error bars indicate 1 SEM. ∗, P < 0.05 vs. WT. LV, left ventricular; RV, right ventricular; ED, end diastole; ES, end systole.


Right ventricular function was assessed in vivo by measurement of right ventricular chamber pressure–volume relationships. RyR2 R176Q/+ mice (n = 7) exhibited smaller end-diastolic volumes: RyR2 R176Q/+, 26.4 ± 0.8 mm3 compared with WT (n = 8) littermates: WT, 31.4 ± 0.9 mm3; P < 0.05, and these values were almost identical to those obtained by MRI (Fig. 3). Moreover, RyR2 R176Q/+ mice had higher right ventricular end-diastolic pressures compared with WT mice: RyR2 R176Q/+, 2.6 ± 0.3 mmHg (1 mmHg = 133 Pa) vs. WT, 1.6 ± 0.2 mmHg; P < 0.05. RyR2 R176Q/+ mice also exhibited nonsignificant trends toward decreased right ventricular stroke volume: RyR2 R176Q/+, 4.1 ± 0.4 μl vs. WT, 4.9 ± 0.6 μl; P = 0.25 and stroke work: RyR2 R176Q/+, 73.2 ± 12.9 mm Hg·μl·min−1 vs. WT, 83.8 ± 13.9 mm Hg·μl·min−1; P = 0.27 compared with WT mice. The lower end-diastolic volume and higher end-diastolic pressure indicate that RyR2 R176Q/+ mice exhibit restrictive ventricular filling.

Fig. 3.
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Fig. 3.

Pressure–volume relationships demonstrate right ventricular diastolic dysfunction in RyR2 R176Q/+ mice. (Upper) End-diastolic pressures were increased in RyR2 R176Q/+ mice (n = 7) compared with WT mice (n = 8). Arrow (Upper Right) denotes the increase in right ventricular diastolic pressure in RyR2 R176Q/+ mice. (Lower) Pressure–volume loops obtained during occlusion of the vena cava demonstrate decreased volumes in the right ventricles of RyR2 R176Q/+ mice.


Isoproterenol Induces Spontaneous Ventricular Ectopy only in RyR2R176Q/+ Mice.

Nine unanesthetized mice (four RyR2 R176Q/+ and five WT) were studied with ECG telemetry. Spontaneous arrhythmias were not observed in any animal before isoproterenol, and ECG intervals in RyR2 R176Q/+ and WT mice were similar at baseline (Table 1). Multiple premature ventricular beats were observed in RyR2 R176Q/+ mice after isoproterenol (Fig. 4 A), whereas no ventricular ectopy was observed in WT mice (Fig. 4 B). Heart rate responses to isoproterenol were somewhat blunted in RyR2 R176Q/+ mice compared with WT mice (RyR2 R176Q/+, 643 ± 16 vs. WT, 676 ± 19; P < 0.05). No sustained ventricular arrhythmias were observed before or after isoproterenol in any mice.

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Table 1.

ECG telemetry data in unanesthetized ambulatory mice


Fig. 4.
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Fig. 4.

Isoproterenol provokes ventricular arrhythmias only in RyR2 R176Q/+ mice. (A) Isoproterenol administration (100 μg i.p.) revealed ventricular trigeminy (every third beat is a premature ventricular beat) in a RyR2 R176Q/+ mouse. (B) No arrhythmias were induced after isoproterenol administration in WT mice. Surface ECG (lead I) during programmed ventricular stimulation showing a short episode of inducible VT at baseline (C) and a longer VT episode after administration of isoproterenol (D) in an RyR2 R176Q/+ mouse. (E) Isoproterenol administration during programmed stimulation increased the number and duration of inducible VT episodes in RyR2 R176Q/+ mice only. (F) Average duration of VT episodes before and after isoproterenol. Error bars indicate 1 SEM. ∗, P < 0.05 baseline vs. isoproterenol.


VT Induction in RyR2R176Q/+ Mice.

Eight animals (four RyR2 R176Q/+ and four WT) completed the electrophysiologic study protocol. Programmed electrical stimulation induced multiple short episodes (<0.5 s) of VT in both groups at baseline (Fig. 4 C), but low-dose isoproterenol increased the number and duration of VT episodes only in RyR2 R176Q/+ mice (Fig. 4 D and E). The average duration of VT was strikingly increased in RyR2 R176Q/+ mice after isoproterenol compared with baseline (baseline, 0.5 ± 0.4 s vs. isoproterenol, 5.1 ± 4.8 s; P < 0.05) and to WT mice in either state (baseline, 0.3 ± 0.1 s, P < 0.05; isoproterenol, 0.5 ± 0.8 s, P < 0.05, Fig. 4 F). There were no differences in electrophysiologic intervals or refractory periods before or after isoproterenol in RyR2 R176Q/+ mice compared with WT (Table 2). Thus, even a low dose of isoproterenol, insufficient to cause a significant increase in heart rate, resulted in significantly more and longer episodes of VT in RyR2 R176Q/+ mice.

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Table 2.

Electrophysiologic data from intracardiac studies in anesthetized mice


To further examine the effect of adrenergic agonists on VT in RyR2 R176Q/+ mice, unanesthetized RyR2 R176Q/+ and WT mice were injected with caffeine (120 mg/kg of body weight) and epinephrine (2 mg/kg of body weight). Two of three RyR2 R176Q/+ mice experienced ventricular ectopy after injection, with one having bidirectional VT (Fig. 5). Ventricular ectopy was not evident in WT mice either before or during acute caffeine/epinephrine treatment.

Fig. 5.
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Fig. 5.

Caffeine and epinephrine injection induce VT in RyR2 R176Q/+ mice only. Tracings in a representative RyR2 R176Q/+ mouse 5 min after injection of caffeine and epinephrine show sinus rhythm (A), ventricular bigeminy (B), and bidirectional VT (C). In B, the upright sinus beats (denoted with asterisks) alternate with premature ventricular beats with an inverted and wider QRS complex. In C, there are two alternating morphologies of QRS complexes: an inverted QRS similar to the ventricular beats seen in B and a wide QRS morphology distinct from the previously observed sinus beats.


Increased Incidence of Spontaneous Ca2+ Oscillations in Single Cardiomyocytes from RyR2R176Q/+ Mice.

One obvious possibility is that mutant RyR2 channels display altered control of Ca2+ release during excitation–contraction. To test this possibility, we measured electrically evoked Ca2+ transients in the absence and presence of isoproterenol (100 nM) in indo-1 AM-loaded ventricular cardiomyocytes isolated from WT and RyR2 R176Q/+ mice. Under control conditions, resting Ca2+ levels, the magnitude of electrically evoked (0.5 Hz) Ca2+ release and maximal response to 10 mM caffeine were comparable (Table 3, which is published as supporting information on the PNAS web site). In addition, stimulation with 100 nM isoproterenol resulted in a similar significant increase in the magnitude of electrically evoked Ca2+ release (Fig. 6 and Table 3). However, a significantly higher incidence of spontaneous, nontriggered Ca2+ oscillations was observed in RyR2 R176Q/+ cardiomyocytes compared with WT myocytes under both control (14.5% vs. 34.7%) and isoproterenol-stimulated conditions (26.3% vs. 57.4%) (Fig. 6; and see Fig. 9C, which is published as supporting information on the PNAS web site). In the presence of isoproterenol (100 nM), RyR2 R176Q/+ cardiomyocytes exhibited a broad spectrum of different types of calcium transients, including normal activity (Fig. 9A), a slowing in the decay of electrically evoked transients (Fig. 9B), nonevoked transients during diastole exhibiting normal kinetics (Figs. 6 B and 9C), and evoked transients exhibiting prolonged, plateau-like durations (Fig. 9D).

Fig. 6.
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Fig. 6.

Increased incidence of isoproterenol-induced, nonevoked Ca2+ oscillations in RyR2 R176Q/+ ventricular myocytes. Representative Ca2+ recordings in the presence of 100 nM isoproterenol (Iso) obtained from indo-1 AM-loaded WT (A) and RyR2R176Q/+ (B) single adult ventricular myocytes. A 10 mM caffeine bolus was applied at the end of the trace to assess sarcoplasmic reticulum calcium content. An expanded time scale of a portion of the electrically evoked transients is shown below each trace. Arrowheads depict the delivery of supramaximal extracellular electrical stimuli. Prominent nonevoked Ca2+ transients (asterisks) were also observed in the RyR2 R176Q/+ myocytes. (C) Average incidence of nonevoked calcium oscillations in WT and RyR2 R176Q/+ myocytes in the presence and absence of 100 nM isoproterenol (ISO).


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Discussion

A common phenotype in CPVT and ARVD2 patients with mutations in RyR2 is the presence of ventricular arrhythmias that can lead to sudden cardiac death (11, 12). Patients with ARVD2 that harbor the R176Q mutation in RyR2 also possess a second RyR2 mutation on the same allele, T2504M (2). In vitro experiments revealed that the R176Q and T2504M mutations, alone or in combination, resulted in altered RyR2 function (8–10). Nevertheless, it was unknown whether the R176Q RyR2 mutation alone is sufficient to cause an increased susceptibility to cardiomyopathy and ventricular arrhythmias in intact animals. Our results demonstrate catecholamine-induced VT in RyR2 R176Q/+ mice that is similar to that observed in patients with ARVD2 and CPVT. Furthermore, altered cardiac function was evident in our RyR R176Q/+ mice, mimicking findings in ARVD2. However, fibrofatty infiltration or fibrosis was not apparent in the right ventricles of RyR2 R176Q/+ mice. We conclude that the R176Q RyR2 mutation alone is sufficient to reduce cardiac function and enhance the incidence of adrenergic-dependent and independent spontaneous Ca2+ oscillations that contribute to catecholamine-induced ventricular arrhythmias.

Although there were no structural abnormalities in hearts of RyR2 R176Q/+ mice up to ≈1 year of age, our data indicate that the RyR2 R176Q mutation contributes to a preclinical cardiomyopathy. This study has measured right ventricular pressure–volume curves in the in vivo mouse heart. The decreased compliance of the right ventricle and decreased systolic function of the left ventricle in RyR2 R176Q/+ mice is most likely caused by a RyR2 calcium leak, leading to increased ventricular myocardial tone. Because ARVD is a progressive disease, it is possible that the modest cardiac dysfunction in RyR R176Q/+ mice may become severe with age. Meticulous evaluation of ventricular function in humans with RyR2 mutations may identify a mild cardiomyopathy in those felt to have structurally normal hearts.

The modest cardiomyopathy seen in our RyR R176Q/+ mice may be more indicative of CPVT rather than ARVD2. It has been postulated that minor cardiac structural abnormalities may be evident in CPVT, but patients that have RyR2 mutations and structural abnormalities are diagnosed as having ARVD2. Although the T2504M RyR2 mutation through unknown mechanisms may serve to further exacerbate this phenotype, our data establish that the R176Q RyR2 mutation is sufficient to drive catecholamine-sensitive arrhythmias and produce a restrictive ventricular filling in the absence of gross structural abnormalities. Our findings also reinforce an emerging concept that “pure” ion channel diseases like CPVT or Brugada syndrome (caused by a loss of sodium channel function) may be associated with minor abnormalities of cardiac structure or contractile function (13, 14).

Recently, Cerrone et al. (15) examined mice with the R4496C mutation in RyR2, which underlies CPVT. These mice did not display any structural abnormalities but demonstrated bidirectional VT in response to adrenergic stress. Cardiac function was not assessed in these mice. We observed bidirectional VT characteristic of CPVT in a RyR R176Q/+ mouse after injection of caffeine and epinephrine. However, the VT episodes induced with programmed stimulation after isoproterenol were not bidirectional but, rather, polymorphic, more similar to those seen in patients with ARVD2. The applicability of our findings of ventricular arrhythmias in RyR R176Q/+ mice to arrhythmias in humans needs further research. Nonetheless, ARVD2 and CPVT, which both arise from RyR2 mutations, may have significant clinical overlap or may represent subtle but distinct variations of a single disorder.

Our results suggest that VT in RyR2 R176Q/+ mice arises from an increase in catecholamine-induced spontaneous calcium release that likely results in the activation of a Ca2+-dependent membrane conductance (e.g., Na+/Ca2+ exchange) and subsequent generation of arrhythmogenic early and delayed after depolarizations. This arrhythmogenic mechanism is analogous to that which is suggested to occur upon sarcoplasmic reticulum calcium overload during digitalis toxicity (16, 17) and in CPVT that results from mutations in the cardiac isoform of calsequestrin (18). The two RyR2 mutations (R176Q and T2504M) have been studied alone and in combination in vitro (9). Alone, each mutation augmented peak Ca2+ release, but demonstrated different Ca2+-release profiles. R176Q had a slower rate of Ca2+ release compared with T2504M, suggesting a complex effect of the double mutation (9). Combined, the mutations increased the sensitivity of Ca2+ release in response to caffeine and lumenal Ca2+ [store overload-induced Ca2+ release (SOICR)] and augmented Ca2+ release compared with WT recombinant RyR2 (8, 10). Our findings in isolated single cardiomyocytes from adult RyR2 R176Q/+ mice are consistent with the SOICR mechanism seen in cells expressing RyR2 containing both mutations.

An alternative mechanism of arrhythmogenesis may result from catecholamine-induced hyperphosphorylation of RyR2 at Ser-2809, which dissociates FKBP12.6 from the channel, causing a diastolic calcium leak (19, 20). To determine whether this leak underlies the abnormal calcium release in RyR2 R176Q/+ cardiomyocytes, we measured RyR2 Ser-2809 phosphorylation in hearts from RyR2 R176Q/+ and WT mice and also after isoproterenol treatment (100 μg i.p.). Although isoproterenol treatment increased RyR2 Ser-2809 phosphorylation, there were no differences between untreated and isoproterenol-treated RyR2 R176Q/+ and WT mice (Fig. 10, which is published as supporting information on the PNAS web site).

We did not observe any histological changes in hearts from RyR2 R176Q/+ mice up to ≈1 year old; however the R176Q RyR2 mutation was sufficient to alter intracellular calcium dynamics, impair ventricular function, and increase susceptibility to catecholamine-induced VT. The phenotype of RyR2 R176Q/+ mice does not precisely correspond to either the CPVT or the ARVD2 phenotypes seen in humans, but demonstrates that the R176Q RyR2 mutation is critical and, possibly, sufficient to confer key aspects of the phenotypes of both diseases and also indicates that these two diseases may have significant overlap.

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Materials and Methods

Generation of Knockin Mice with the RyR2 R176Q Mutation.

A genomic clone containing exons 7 and 8 of the mouse RyR2 was isolated from a 129/SvJ λKO-1 library by using the recombination cloning system in ref. 21. The R176Q mutation, along with a new RsrII restriction site, was introduced into exon 8 of the RyR2 gene. A cassette containing a lox P-flanked Neo R gene expressed from the phosphoglycerate kinase promoter (PGK-Neo R) and a Tet R gene was cloned in the unique BlpI site from intron 8 of our genomic clone to obtain the final targeting vector (RyR2 R176QNeo). The targeting vector was linearized with PmeI and electroporated into AB2.2 129Sv/J ES cells. DNA was isolated from G418 and gancyclovir double-resistant clones and subjected to Southern blot analysis. A clone found to include the homologous targeted integrand was verified by RsrII restriction digestion and direct sequencing and injected into C57BL/6 blastocysts, giving rise to germ-line transmission resulting in F1 RyR2 R176QNeo/+ mice. These mice were mated onto Tg (EIIA-Cre) mice (a gift from Graeme Mardon, Baylor College of Medicine) to remove the floxed PGK-Neo R/Tet R cassette. RyR2 R176Q/+ mice were backcrossed three times on C57/Bl6 background. For genotyping, genomic DNA was prepared from tails and subjected to PCR and digestion with RsrII.

Histology.

Hearts from male RyR2 R176Q/+ mice (n = 7) and WT control littermates (n = 7), aged 16–22 weeks or 51–57 weeks, were fixed with 10% formalin. The ventricular component of each heart was divided into three cross-sections, submitted in a single paraffin block, and processed by routine histologic techniques. A complete, full-circumferential section of the middle slice of the ventricles, which consistently included the midportion of the two left ventricular papillary muscles, was selected for morphometric analysis. In some hearts, partial collapse of the right ventricular freewall necessitated realignment of the collapsed segment before analysis. In these cases, the contour of the affected freewall segment was redrawn. Measurements of the individual areas of the left and right ventricular chambers from each heart were made by using a computer-assisted digitizing system with software from Bioquant (Nashville, TN).

Noninvasive Cardiac Function.

Cardiac function was analyzed by using Doppler ultrasound (Fig. 8), MRI, and cardiac catheterization. For MRI, heterozygous RyR2 R176Q/+ (n = 6) and WT (n = 6) mice were initially anesthetized with 4–5% isoflurane (mixed with oxygen) and maintained with 1–2% isoflurane during imaging. An animal-monitoring system (SA Instruments, Stony Brook, NY) was used to monitor the mouse’s ECG, respiratory rate, and body temperature. Respiratory- and cardiac-gated images were acquired at end-diastole and end-systole by using a 9.4T, Bruker Avance, 21-cm-bore horizontal scanner. The imaging parameters to acquire cardiac- and respiratory-gated spin echo images were as follows: repetition time (TR), 800 ms; echo time (TE), 10 ms; field of view, 3.0 cm; number of slices, 10; slice thickness, 1.0 mm; matrix, 256 × 256; and number of averages, 1. The multislice scan was performed in the axial orientation to visualize the left and right ventricles, and data were analyzed by using Amira 3D image processing software (Mercury Computer Systems, Chelmsford, MA). The areas representing the left and right ventricular space in each slice were summated in the amount of pixels. The volume size of the space was calculated by using the known volume size of each pixel (30.0 mm/256 × 30.0 mm/256 × 1.0 mm).

For right ventricular catheterization, RyR2 R176Q/+ (n = 7) and WT (n = 8) mice were anesthetized with 1.5% isoflurane (mixed with oxygen) and maintained at 37°C by a heating pad during the experiment. A 1.4F high-fidelity micromanometer catheter (Millar Instruments) was advanced via the right jugular vein into the right ventricle. Pressure–volume relationships were measured under baseline conditions and during transient occlusion of the inferior vena cava. End-diastolic pressure–volume relationships were fitted by using the monoexponential equation. An IOX data acquisition system (Emka Technologies, Falls Church, VA) was used to analyze pressure–volume relationships.

ECG Telemetry.

Nine animals (four RyR2 R176Q/+ and five WT) were studied with ECG telemetry according to published methods (22, 23). Briefly, transmitters (Data Sciences International, St. Paul, MN) were implanted in the abdominal cavity with s.c. electrodes in a lead I configuration. Telemetry was recorded >24–72 h after surgery in ambulatory, unanesthetized mice for 30-min intervals at baseline and after the injection of isoproterenol 100 μg i.p. A subset of telemetered RyR2 R176Q/+ and WT mice (n = 3 in each group) were injected with epinephrine (2 mg/kg of body weight i.p.) and caffeine (120 mg/kg of body weight i.p.) and monitored for 10 min. Data collection was performed by using Dataquest software, and off-line data analyses were performed by using Physiostat ECG analysis software, version 3.1 (Data Sciences International).

Intracardiac Electrophysiology Studies.

RyR2 R176Q/+ (n = 4) and WT (n = 4) mice were anesthetized with pentobarbital (0.07 mg/g of body weight i.p.). Surface ECG (lead I) signals were obtained from s.c. 27-gauge needles placed in each forelimb. The ECG channels were amplified (0.1 mV/cm) and filtered between 0.05 and 400 Hz. An octapolar 2F electrode catheter (CIBer cath; NuMED, Hopkinton, NY) was placed in the right atrium and right ventricle. Bipolar electrogram recordings were obtained from the right atrium, right ventricle, and His positions. Signals were amplified and filtered between 40 and 400 Hz at a 200- to 400-mm/s speed. Bipolar pacing was performed by using a programmable stimulator (Medtronic 2356) modified by the manufacturer to deliver coupling intervals as short as 10 ms. Pacing threshold (in milliamperes) was determined for each pacing site, and stimulation was performed for 1.0- to 2.0-ms pulse width at twice the diastolic-capture threshold. Standard clinical electrophysiologic pacing protocols were used to determine all basic electrophysiologic parameters. Ventricular effective refractory period was determined at three drive cycle lengths. Single, double, and triple extrastimuli were delivered at three drive cycle lengths to determine inducibility of VT. After baseline measurements were completed, isoproterenol was administered (100 μg i.p.) and the protocols repeated to assess the effects on conduction and refractoriness.

Intracellular Ca2+ Measurements.

Single ventricular myocytes were isolated from the hearts of 8- to 16-week-old WT and RyR2 R176Q/+ mice by using standard enzymatic dissociation and plated on laminin-coated dishes as described by the Alliance for Cellular Signaling (AfCS Procedure Protocol ID PP00000 125) (24). Myocytes were loaded with 6 μM indo-1 AM (Molecular Probes) for 60 min at 37°C and superfused with a Ringer’s solution containing 146 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes (pH 7.4). Indo-1-loaded myocytes were excited at 350 nm by using a DeltaRam illumination system (Photon Technology International, Lawrenceville, NJ). Fluorescence emission at 405 and 485 nm was collected at 100 Hz by using a photomultiplier detection system and represented as the ratio (R) of F405/F485. For measurements of electrically evoked calcium release, myocytes were stimulated (8 V, 20 ms, 0.5 Hz for 60 s) by using an extracellular electrode placed close to the cell of interest in the absence and presence of 100 nM isoproterenol. A 10 mM caffeine bolus after the pulse train was used to assess sarcoplasmic reticulum Ca2+ content.

Statistical Analyses.

Electrophysiologic and cardiac function and intracellular Ca2+ measurements were compared between RyR2 R176Q/+ and WT animals with Student’s t test. Effects of isoproterenol on ECG parameters were compared with a paired t test. A two-tailed P value of <0.05 was considered significant. For the data presented in Fig. 6 C, statistical significance was determined by using a χ2 test. All procedures were approved by the respective institution’s animal care committee.

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Acknowledgments

We thank Lingyun Hu and Fredalina Pieri for technical assistance with the MRI and telemetry data, respectively. This work was supported, in part, by U.S. Public Health Service Grants AR41802, HL070250, HL046681, AG17899, HL22512, and AR44657. P.J.K. was supported by a Vanderbilt University School of Medicine Clinician Scientist Award, M.E.A. is an Established Investigator of the American Heart Association, and X.H.T.W. was supported by an American Heart Association Scientist Development Grant.

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Footnotes

  • §§To whom correspondence should be addressed at:
    Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
    E-mail: susanh{at}bcm.tmc.edu

  • Author contributions: P.J.K., B.M.M., S.A.G., M.G.C., S.S., C.I.D., X.H.T.W., R.G.P., R.T.D., M.E.A., and S.L.H. designed research; P.J.K., B.M.M., S.A.G., M.G.C., W.Z., S.S., D.L.K., C.I.D., M.D.B., X.H.T.W., R.G.P., G.E.T., R.T.D., and M.E.A. performed research; M.G.C. and X.H.T.W. contributed new reagents/analytic tools; P.J.K., B.M.M., S.A.G., D.L.K., C.I.D., M.D.B., X.H.T.W., R.G.P., D.M.R., G.E.T., R.T.D., M.E.A., and S.L.H. analyzed data; and P.J.K., B.M.M., D.M.R., R.T.D., M.E.A., and S.L.H. wrote the paper.

  • Conflict of interest statement: No conflicts declared.

  • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviations:
    ARVD,
    arrhythmogenic right ventricular dysplasia;
    CPVT,
    catecholaminergic polymorphic ventricular tachycardia;
    RyR 2,
    ryanodine receptor 2;
    VT,
    ventricular tachycardia.
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