No Contribution of IP₃-R(2) to Disease Phenotype in Models of Dilated Cardiomyopathy or Pressure Overload Hypertrophy

Abstract

Background

We investigated the contribution of inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃, IP₃) receptors (IP₃-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20 like kinase and dominant negative phosphatidylinositol-3-kinase in heart (Mst1 x dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of IP₃-R(2) and heightened generation of Ins(1,4,5)P₃. Similar increases in Ins(1,4,5)P₃ and IP₃-R(2) are caused by thoracic aortic constriction (TAC).

Methods and Results

To evaluate the contribution of IP₃-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP₃-R (IP₃-R(2)−/−) had been deleted (DCM-2Tg x IP₃-R(2) −/−) and TAC was performed on IP₃-R(2) −/−mice. Hearts from DCM-2Tg mice and DCM-2Tg x IP₃-R(2) −/− were similar in terms of chamber dilatation, atrial enlargement and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP₃-R(2). Deletion of IP₃-R(2) did not alter the progression of heart failure, as DCM-2Tg mice with and without IP₃-R(2) had similarly reduced contractility, increased lung congestion, atrial thrombus and both strains died between 10 and 12 weeks of age. Loss of IP₃-R(2) did not alter the progression of hypertrophy following TAC.

Conclusions

We conclude that IP₃-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy despite increased expression and heightened generation of the ligand, Ins(1,4,5)P₃.

The failing myocardium from humans and experimental animals commonly shows increased expression of the receptors for the Ca²⁺-regulating second messenger Ins(1,4,5)P₃ (IP₃-receptors, IP₃-R), often along with a decrease in ryanodine receptors, the master regulators of Ca²⁺ within the myocardium ¹. The importance, or otherwise, of this has been debated. The altered relative expression levels of the intracellular Ca²⁺ channels might reflect a degree of de-differentiation of the failing ventricular myocytes, with no major functional implications. Alternatively, increased IP₃-R might contribute to, or ameliorate, the progression of heart failure. IP₃-R have been suggested to contribute to cardiomyocyte hypertrophy ^2,3, to chamber dilatation ⁴ and to arrhythmogenesis ⁵, but there is little agreement between laboratories in relation to any of these claims.

Under physiological conditions cardiomyocytes from all species studied show little or no change in Ins(1,4,5)P₃ in response to the activation of appropriately coupled receptors ⁶, and in addition the expression of IP₃-R is low ⁷. These findings argue against a strong involvement of IP₃-R under physiological conditions. However, our earlier and recent studies show that generation of Ins(1,4,5)P₃ is markedly heightened under such pathological conditions as atrial dilatation in humans, mice with dilated cardiomyopathy ⁴ and in hypertrophied mouse ventricle following chronic pressure overload ⁸. Furthermore, we have observed heightened expression of phospholipase Cβ1b (PLCβ1b), the enzyme responsible for generation of Ins(1,4,5)P₃ in myocytes, from failing human, mouse and sheep myocardium ⁴ (sheep data not shown). All of these findings suggest that Ins(1,4,5)P₃ may be of heightened importance in failing myocardium, especially given the likelihood of increased IP₃-R expression ^7,9.

We undertook an investigation of possible pathological roles of IP₃-R using a mouse model with severe dilated cardiomyopathy (DCM) and clear evidence of conduction block (DCM-2Tg) as well as a well characterized model of pressure overload hypertrophy, induced by trans-aortic constriction (TAC) ⁸. Both of these murine model show increased generation of Ins(1,4,5)P₃ ^4,8 and increased expression of IP₃-R(2), the IP₃-R subtype expressed in working cardiomyocytes. To examine possible contributions of the heightened Ins(1,4,5)P₃ and IP₃-R(2) to hypertrophy and dilatation, we performed TAC on IP₃-R(2) −/− mice and crossed the DCM-2Tg with IP₃-R(2) −/− mice and examined the effect on progression of disease in both scenarios.

Methods

Experimental Animals

All experiments were approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee. DCM-2Tg model. Cardiac-specific Mst1 (line no. 28; C57BL/6 background) and dnPI3K (FVB/N) transgenic mice were generated and genotyped as described previously ^10,11. IP₃-R(2) −/− mice were generated on a C57BL6 background, as described ⁵. Male heterozygous Mst1-Tg (Mst1^+/−) were genetically crossed with dnPI3K^+/− and IP₃-R(2) −/− to generate DCM-2Tg mice (Mst1^+/−dnPI3K^+/−, Mst1xdnPI3K-2Tg, DCM-2Tg) and DCM-2Tg-KO mice (Mst1^+/−/dnPI3K^+/−/IP₃-R(2) −/−) ¹². The crossed strain (Mst1xdnPI3K2-Tg) was chosen for these studies because, unlike the Mst1-Tg, they show severe conduction block and we were interested in possible atrial actions of IP₃-R(2). All mice were bred on the same mixed genetic background (C57BL/6/FVB/N).

Transverse aortic constriction

Two-month-old male mice were anesthetized with ketamine /xylazine, and underwent a sham operation or were subjected to pressure overload induced by thoracic aortic constriction (TAC) as previously described ¹³. Echocardiography measurements were performed before surgery, 1 and 4 weeks post-TAC. The pressure gradients generated by aortic banding were measured by introducing high fidelity pressure transducers into the left and right common carotids. Pressure gradients in IP₃-R(2) −/− and WT littermates were similar (Supplemental Figure 1A). Hearts were collected and morphometrics evaluated 4 weeks post-TAC.

Transthoracic echocardiography and electrocardiography (ECG)

Anesthesia was maintained with 1.7% isoflurane. Echocardiography was performed using a Philips iE33 ultrasound machine and a 15 MHz linear-transducer. ECG recordings were measured using the Powerlab System and BioAmp (ADInstruments). Data were measured digitally using ADInstruments (Chart 5 Pro ECG analysis module). Details are provided in the online supplement.

Measurement of Ins(1,4,5)P₃ and its metabolites in atria and ventricles (PLC activation)

Tissues were labeled with [³H]inositol and subsequently stimulated with norepinephrine (50 μmol/L). [³H]Ins(1,4,5)P₃ and its metabolites were extracted and quantified by HPLC, as described previously ^4,14. Details are provided in the online supplement.

Measurement of mRNA and protein expression in atria and ventricles

RNA was extracted using RNEasy kits from Qiagen or Trizol reagent (Invitrogen) according to the manufacturer’s instructions, and reverse transcribed using Superscript III (Invitrogen). Quantitative real-time qRT-PCR with SybrGreen (Invitrogen) reagent was performed on an Applied Biosciences 7500 Fast Real Time PCR System. Primer sequences are provided in the online supplement. Values are expressed as the 2^−ΔCt value relative to GAPDH in each sample ¹⁵. All qRT-PCR experiments were performed in triplicate on triplicate or quadruplicate samples. RNA dot-blot analyses were performed as previously described ¹⁶. Protein methods are outlines in the online supplement.

Data evaluation

For experiments involving two groups (WT and DCM or sham and TAC), or where n=3, data were analyzed using a Mann-Whitney Rank Sum Test. For experiments involving 4 groups (WT, IP₃-R(2) −/−, DCM, DCM x IP₃-R(2) −/− or Sham, sh. IP₃-R(2) −/−, TAC, TAC IP₃-R(2) −/−) a 2-Way ANOVA was used to assess the effect of TAC or DCM, the IP₃-R(2) status and the interaction between the two (Sigma Stat). Where repeated measures were involved (Echo measurements after TAC) a Repeated Measures 2-way ANOVA was used. All pairwise multiple comparisons used a Holm-Sidak test post hoc, except where normality tests failed and a Rank Sum test was used (Sigma Stat).

Results

Hearts from DCM-2Tg mice or TAC mice have heightened expression of IP₃-R(2) and heightened generation of Ins(1,4,5)P₃

The type 2 IP₃-R is expressed in working cardiomyocytes and we measured expression in hearts from DCM-2Tg mice that have dilated cardiomyopathy as well as from mice that have undergone TAC to induce pressure overload hypertrophy. The DCM-2Tg mice had heightened in IP₃-R(2) expression in atria and left ventricles (Figure 1A). Loss of IP₃-R(2)in IP₃-R(2) −/−mice, did not result in altered expression of either of the other IP₃-R subtypes, IP₃-R(1) or IP₃-R(3) (Supplemental Figure 1). IP₃-R(2) expression also was substantially elevated 4 weeks after TAC (Figure 1B&C).

Figure 1. Hearts of DCM-2Tg mice and TAC mice have heightened IP₃-R(2) expression and InsP generation.

A. Expression of IP₃-R(2) in atria and ventricles from DCM-2Tg mice and littermate controls (WT). Values shown are IP₃-R(2) mRNA expressed as fold change, mean ±SEM, n=5–7. B. Expression of IP₃-R(2) (mRNA relative to GAPDH) in left ventricle of mice 4 weeks after TAC. C. Expression of IP₃-R(2) at the protein level 4 weeks after TAC. The experiment was performed 3 times with similar results. D. Generation of [³H]Ins(1.4.5)P₃ and its metabolites in atria and ventricles from WT and DCM-2Tg mice. Values shown are [³H]inositol phosphates, CPM/mg of tissue, mean ± SEM, n=5–7, * p<0.05 relative to WT or sham operated control (Mann-Whitney, Rank Sum Test).

We have previously reported that TAC induced hypertrophy leads to heightened InsP responses in mouse hearts resulting in increased generation of Ins(1,4,5)P₃ ⁸. In the current study we assessed Ins(1,4,5)P₃ generation in atria and ventricles from DCM-2Tg and littermate WT mice. This was accomplished by labeling the inositol phospholipids with [³H]inositol and subsequently measuring the generation of [³H]-labeled Ins(1,4,5)P₃ and its metabolites as a measure of total [³H]Ins(1,4,5)P₃ generation. As described previously ⁴, atria and ventricles from WT mice have low levels of [³H]InsPs after 20 min treatment with norepinephrine (50 μM), indicating minimal generation of Ins(1,4,5)P₃. Responses were substantially heightened in atria and ventricles from DCM-2Tg mice reflecting increased Ins(1,4,5)P₃ production (Figure 1D).

Deletion of IP₃-R(2) does not influence heart size in DCM-2Tg mice or TAC mice

DCM-2Tg mice develop dilated cardiomyopathy indicated by chamber dilatation, LV wall thinning and decreased contractile function ¹². We argued that heightened generation of Ins(1,4,5)P₃, together with heightened expression of IP₃-R(2), might contribute substantially to the DCM phenotype in these animals. To test this possibility, we examined the phenotype of the DCM-2Tg mice in which IP₃-R(2) had been deleted (DCM-2TgxIP₃-R(2) −/−), and compared this with DCM mice expressing the endogenous complement of IP₃-R(2) (DCM-2Tg). As reported previously ¹², DCM mice exhibited severe LV dilatation (Supplemental Figure 2B, Table 1), increased atrial size, and ventricular wall thinning, compared with WT mice (Table 1). There was no hypertrophy observed in the DCM-2Tg hearts. DCM-2Tg mice with IP₃-R(2) deleted showed a phenotype indistinguishable from that of DCM-2Tg mice (Table 1, 2).

Table 1.

Echocardiographic parameters in DCM-2Tg mice and the effect of deletion of IP₃-R(2)

	WT	IP3-R(2) −/−	DCM-2Tg	DCM-2Tg x IP3-R(2) −/−	p DCM	p IP3-R(2) −/−	p int.
	(7)	(5)	(6)	(12)
Gender males/females	5/2	4/1	2/4	5/7
Age (weeks)	9.1 ± 1.4	7.2 ± 0.5	8.0 ± 0.1	8.8 ± 0.7
HR, bpm (echo)	571 ± 30	628 ± 48	490 ± 34	507 ± 20	ns	ns	ns
LVPWs mm	1.3 ± 0.10	1.2 ± 0.05	0.75 ± 0.05	0.8 ± 0.05	<0.001	0.619	0.943
LVPWd mm	0.92 ± 0.09	0.94 ± 0.06	0.66 ± 0.04	0.68 ± 0.05	<0.001	0.516	0.201
LVIDs mm	1.97 ± 0.18	2.2 ± 0.26	3.69 ± 0.14	3.7± 0.18	<0.001	0.324	0.203
LVIDd mm	3.5 ± 0.17	3.4 ± 0.2	4.2 ± 0.18	4.3 ± 0.16	<0.001	0.567	0.660
IVSs mm	1.53 ± 0.11	1.4 ± 0.12	0.79 ± 0.06	0.87 ± 0.06	<0.001	0.762	0.181
IVSd mm	0.96 ± 0.07	1.02 ± 0.04	0.68 ± 0.04	0.71 ± 0.04	<0.001	0.373	0.830
LA area mm2	7.6 ± 0.58	7.24 ± 0.9	21.7 ± 2.3	19.68 ± 1.6	<0.001	0.593	0.637

Values are mean ± SEM. The number of animals is indicated in brackets. Data were analyzed by 2 way ANOVA and p values shown are for the effect of DCM, effect of IP₃-R(2) −/− and the interaction (int.) between DCM and IP₃-R(2) −/−. ns = not significant.

Table 2.

Morphometric parameters in DCM-2Tg mice and the effect of deletion of IP₃-R(2)

	WT	IP3-(2) −/−	DCM-2Tg	DCM-2Tg x IP3-R(2) −/−	p DCM	p IP3-R(2) −/−	p Int.
	(6)	(6)	(12)	(18)
Gender (m/f)	4/2	4/2	5/7	8/10
Age, weeks	13 ± 0.58	10 ± 0.75	10.5 ± 1.3	11.8 ± 1.4
Ventricle/tibia, mg/mm	7.05 ± 0.17	6.85 ± 0.41	6.41 ± 0.41	6.47 ± 0.30	ns	ns	ns
Atria/tibia, mg/mm	0.39 ± 0.09	0.37 ± 0.05	0.94 ± 0.2	0.88 ± 0.11	<0.001	0.486	0.294
Body wt, g	28.2± 2.4	31.4 ± 3.8	23.8 ± 1.6	26.8 ± 1.3	ns	ns	ns

Values are mean ± SEM. The number of animals is indicated in brackets. Data were analyzed by 2 way ANOVA and p values shown are for the effect of DCM, effect of IP₃-R(2) −/− and the interaction (int.) between DCM and IP₃-R(2) −/−. ns = not significant.

In contrast to the DCM-2Tg model, mice subjected to TAC develop ventricular hypertrophy indicated by increased LV/body weight ratio and increased ventricular wall thickness. Deletion of IP₃-R(2) did not alter the cardiac phenotype either in TAC mice or in sham operated controls. Measurements were made both early (1 week) and late (4 weeks after TAC) and no effect of IP₃-R(2) deletion were observed at either time point Figure 2).

Figure 2. Deletion of IP₃-R(2) does not alter hypertrophic responses at 1 or 4 weeks after TAC.

Echocardiographic parameters measured in IP₃-R(2) −/− mice and littermate controls (WT, +/+), pre-TAC and at 1 week and 4 weeks after TAC. Values shown are mean +/− SEM, n=6 for sham groups, 13 for +/+ TAC and 15 for −/− TAC. Analyses used a 2-Way Repeated Measures ANOVA, as described in the Methods section. Significant effect of TAC, relative to sham, †† p<0.001, ‡ p<0.01, † p<0.05. There was no significant effect of IP₃-R(2) −/− on any parameter irrespective of the TAC status.

Deletion of IP₃-R(2) does not alter gene expression profiles in DCM-2Tg mice or TAC mice

DCM-2Tg mice show alterations in ventricular gene expression, reflecting disease progression¹². We examined transcriptional responses in DCM mice with and without their complement of IP₃-R(2). As shown in Figure 3A, DCM in the DCM-2Tg was associated with increased expression of atrial natriuretic peptide (ANP) and α-skeletal actin (α-Sk. actin). Deletion of IP₃-R(2) did not alter these expression changes. As reported previously ⁸, TAC induced changes in ‘hypertrophic’ gene expression profiles, ANP, α-myosin heavy chain (MHC), β-MHC and α-skeletal actin, and these were not altered by deletion of IP₃-R(2) (Figure 3C&D).

Figure 3. Lack of effect of IP₃-R(2).

−/− on transcriptional changes in DCM-2Tg mice or TAC mice.

A. Expression of hypertrophic marker genes in left ventricle from the DCM-2Tg and WT mice with and without IP₃-R(2). Values shown are mRNA/GAPDH, fold change, mean ± SEM (n=4–6). B. Expression of modulatory calcineurin-interacting protein (MCIP) in LV from sham-operated and TAC mice 4 weeks after surgery (n=6). C. Dot blots showing mRNA expression in left ventricle from sham-operated and TAC mice with (+/+) or without IP₃-R(2) (−/−). D. Quantification of the data shown in C. Values shown are mRNA/GAPDH, fold change, mean +/− SEM, n=3. †† p<0.001, ‡ p<0.01, † p<0.05 relative to WT or sham. There was no significant effect of IP₃-R(2) −/− on any parameter irrespective of the DCM-2Tg or TAC status and there was no interaction detected between IP₃-R(2) −/− and DCM-2Tg or TAC.

Ins(1,4,5)P₃/IP₃-R(2) signaling has been suggested to contribute to hypertrophy by generating the Ca²⁺ required to activate calcinuerin, which in turn dephosphorylates the NFAT family of transcription factors culminating in altered gene expression profiles ¹⁷. Calcineurin is known to be activated by TAC and we examined whether deletion of IP₃-R(2) altered calcineurin signaling after TAC. Modulatory calcineurin-interacting protein (MCIP) is a direct tranascriptional target of calciuenrin and we measured MCIP as an index of calcineurin signaling. As shown in Figure 3B, MCIP expression was increased at 4 weeks following TAC, but deletion of IP₃-R(2) did not influence expression either in TAC or sham operated mice.

Deletion of IP₃-R(2) does not alter ECG profiles in DCM-2Tg mice

We next examined whether deletion of IP₃-R(2) influenced electrophysiological changes we have previously found to be associated with heart failure in the DCM model ¹². No arrhythmias were observed over the time periods studied. Relative to WT littermates, ECG profiles from DCM-2Tg mice showed prolonged P-R interval, prolonged Q-T interval and reduced R amplitude. R-R interval, QRS interval and ST height were not significantly altered in the DCM mice. Deletion of IP₃-R(2) did not influence these changes and data obtained from DCM-2Tg and DCM-2TgxIP₃-R(2) −/− were not different in terms of any of these parameters. Deletion of the IP₃-R(2) also did not alter ECG profiles in the absence of DCM (Supplemental Figure 2C, Table 3).

Table 3.

ECG parameters in DCM-2Tg mice and the effect of deletion of IP₃-R(2)

	WT	IP3-R(2) −/−	DCM-2Tg	DCM-2Tg x IP3-R(2) −/−	p DCM	p IP3-R(2) −/−	p Int.
	(8)	(5)	(8)	(13)
Gender (m/f)	5/3	3/2	4/4	5/8
Age (weeks)	9.3 ± 1	10 ± 3	9.2 ± 0.8	11.4 ± 1.6
R amp., mV	1.26 ± 0.2	1.15 ± 0.16	0.66 ± 0.09	0.86 ± 0.11	<0.001	0.252	0.950
P amp, mV	0.13 ± 0.01	0.16 ± 0.01	0.057 ±0.01	0.048 ± 0.01	<0.001	0.374	0.500
P-R int. ms	37 ± 1	35 ± 2	54 ± 4	56 ± 3	<0.001	0.825	0.485
Q-T int. ms	17 ± 1.5	14 ± 1	42± 5	39 ± 3	<0.001	0.482	0.239
QRS int. ms	8 ± 1	8 ± 1	10 ± 1	10 ± 1	ns	ns	ns
R-R int. ms	112 ± 7	111 ± 4	119 ± 5	124 ± 2	ns	ns	ns

Values are mean ± SEM. The number of animals is indicated in brackets. Data were analyzed by 2 way ANOVA and p values shown are for the effect of DCM, effect of IP₃-R(2) −/− and the interaction (int.) between DCM and IP₃-R(2) −/−. ns = not significant.

Deletion of IP₃-R(2) does not alter functional parameters in DCM-2Tg mice or in TAC mice

DCM-2Tg mice die prematurely by 10–12 weeks of age. Deletion of IP₃-R(2) did not alter the death rate in this strain (Figure 4A). DCM-2Tg mice showed a high incidence of pleural effusion, lung congestion and atrial thrombus, consistent with severe heart failure. Deletion of IP₃-R(2) did not alter any of these parameters (Figure 4B). Contractility, measured as fractional shortening by echocardiography, was substantially reduced in the DCM-2Tg mice, but was not influenced by deletion of IP₃-R(2) (Figure 4C, Table 1). Lung congestion was similar in DCM-2Tg with and without their complement of IP₃-R(2) (Figure 4D). Thus, IP₃-R(2) do not contribute substantially to the heart failure phenotype in this model.

Figure 4. Deletion of IP₃-R(2) does not alter the phenotype of the DCM-2Tg mice or TAC mice.

A. Kaplan-Meier survival curves of DCM-2Tg mice, 10 total, (black symbols) and DCM-2TgxIP₃-R(2) −/− mice, 10 total (red symbols). There was no detectable difference between death rates. B. Incidence of pleural effusion, LA and RA thrombus in DCM-2Tg mice with (+/+) and without (−/−) their complement of IP₃-R(2). Number of positive animals relative to the total animal number is shown on the bars. The DCM-2Tg +/+ group contained 7/13 females and the −/− group 10/19 females. None of the values was significantly different between the two groups. C. Fractional shortening, measured by echocardiography, in DCM-2Tg mice and DCM-2Tg x IP₃-R(2) −/− mice. Values shown are mean ± SEM (n=6 for the DCM-2Tg group and 12 for the DCM-2Tg x IP₃-R(2) −/− group). †† p<0.001 relative to WT or IP₃-R(2) −/− mice. D. Lung wt. relative to tibia length (mg/mm) in DCM-2Tg and DCM-2Tg x IP₃-R(2) −/− mice. Values shown are mean ± SEM (n=11 for the DCM-2Tg group and 14 for the DCM-2Tg x IP₃-R(2) −/− group). E. Fractional shortening, measured by echocardiography in sham-operated and TAC mice, before and 1 and 4 weeks after surgery. Values shown are mean +/− SEM, n=6 for the sham operated groups, 13 for +/+ TAC and 15 for −/− TAC. F. LV weight relative to body weight in sham-operated and TAC mice 4 weeks after surgery. Values shown are mean ± SEM, n=6 for sham groups, 13 for +/+ TAC and 15 for −/− TAC. †† p<0.001 relative to sham. There was no significant effect of IP₃-R(2) −/− on any parameter irrespective of the DCM-2Tg or TAC status and there was no interaction detected between IP₃-R(2) −/− and DCM-2Tg or TAC. Except for the survival study (A), all measurements on DCM-2Tg mice were made between 6 and 8 weeks of age.

TAC resulted in a progressive decrease in fractional shortening that was not altered by IP₃-R(2) deletion (Figure 4E). Similarly increases in left ventricular weight were similar in WT and IP₃-R(2) −/− mice following TAC (Figure 4F).

Discussion

Heart failure in humans and experimental animals has repeatedly been reported to be associated with increased ventricular expression of IP₃-R(2) in the left ventricle ^7,18,19. Increased atrial expression has been reported in valvular heart disease and atrial fibrillation ⁹. Furthermore, increased IP₃-R(2) expression has been reported in neonatal rat cardiomyocytes undergoing hypertrophy ². In some cases the increased IP₃-R(2) expression is paralleled by a lowering of ryanodine receptor expression ^{7, 18}. These findings might suggest that IP₃-R(2) are of increased importance in hypertrophic and failing myocardium. It has been suggested that the increased IP₃-R activity contributes to pathology by promoting hypertrophic growth of the cardiomyocytes ^{3, 20} or that the increased IP₃-R might contribute to arrhythmia by perturbing Ca²⁺ responses close to sarcolemmal Ca²⁺ channels and/or the Na⁺/Ca²⁺ exchanger ^{5, 21}. Alternatively, the changes might reflect a loss of muscle phenotype as the cardiomyocytes undergo the transition to failure and have little functional consequence. We recently reported that dilated atrial tissues from patients with valvular heart disease have substantially heightened generation of Ins(1,4,5)P₃, and furthermore that generation correlated with atrial volume, suggesting a relationship to atrial dilatation. Similar observations were made in the dilated atria from hearts of mice overexpressing Mst1, where phospholipase C activity correlated with atrial weight ⁴.

Transgenic mice expressing Mst1 in heart have been reported previously to have dilated cardiomyopathy ¹⁰, and the phenotype is exacerbated by co-expression of dominant negative PI3-Kinase ¹². The crossed strain (Mst1xdn-PI3K-2Tg; DCM-2Tg) has a worsened heart failure phenotype compared with Mst1-Tg, and in addition, shows clear evidence of conduction block, reflected by increased P-R interval ¹² (Table 3). Both the Mst1-Tg and Mst1xdn-PI3K-2Tg strains express heightened levels of IP₃-R(2) in atria and ventricles and additionally both have substantially increased Ins(1,4,5)P₃ generation (Figure 1) ⁴. We reasoned that the heightened Ins(1,4,5)P₃/IP₃-R axis in this mouse strain provided an excellent opportunity to evaluate its contribution to the DCM phenotype. The crossed strain (Mst1xdn-PI3K-2Tg; DCM-2Tg) was chosen for these studies because we were interested in a possible contribution of Ins(1,4,5)P₃ and IP₃-R(2) to conduction block, as well as to chamber dilatation and heart failure. The DCM model involves chamber dilatation, possibly related to enhanced cardiomyocyte apoptosis, without cardiomyocyte hypertrophy ¹⁰. In addition to a contribution to heart failure, IP₃-R have been suggested to contribute to hypertrophic signaling pathways, by supplying Ca²⁺ to activate the calcineurin/NFAT pathway ³, or by activating CaMKII localized close to the nuclear membrane ²⁰. To evaluate possible contributions of the Ins(1,4,5)P₃/IP₃-R(2) axis to hypertrophy we used a well-established pressure overload model ²².

When we compared the DCM-2Tg mice with DCM-2Tg mice lacking IP₃-R(2), we found no difference in the extent of dilatation, chamber size or contractile dysfunction. Indices of heart failure, lung congestion, atrial thrombus and pleural effusion also were similar between the two experimental groups. Life span also was similar in the two groups (Figure 4A), showing that elimination of IP₃-R(2) did not slow disease progression. ECG studies on DCM mice revealed increased P-R interval indicative of conduction block and long Q-T interval, reflecting prolonged action potential duration Neither of these perturbations was altered by deletion of IP₃-R(2) (Table 3). Hypertrophic responses to pressure overload also were not altered by removal of IP₃-R(2). At either 1 week or 4 weeks after TAC, WT and IP₃-R(2) −/− mice showed similar changes in ventricular dimensions and ventricular weight (Figure 2, 4F). Failure to detect any effect of deleting IP₃-R(2) on any of the pathological changes observed in either DCM-2Tg mice or in mice subjected to TAC argues strongly that Ins(1,4,5)P₃ and its receptors do not make a major contribution, either positively or negatively, to hypertrophy, chamber dilatation or conduction block in these models.

Heightened IP₃-R expression has been a common finding in a range of cardiac pathologies. Heightened left ventricular expression has been reported in failing human heart ^{1, 23}, in a rabbit model of non-ischemic heart failure ¹⁸ as well as in the murine DCM model used in this study and following TAC (Figure 1). IP₃-R expression/activity is also increased in atrial tissue from patients with atrial fibrillation associated with valvular heart disease ^{9, 24}. In the latter study, the increased IP₃-R expression reversed following cardioversion to sinus rhythm, suggesting a contribution of IP₃-R to AF in this patient group. However, the expression of a number of ion channels and transporters showed a similar reversion with return to sinus rhythm, and the observed changes might be a consequence of, rather than a contributor to AF, as suggested by another study ²⁵. In contrast to IP₃-R expression studies, assessment of changes in the generation of Ins(1,4,5)P₃ in heart disease states has been limited. Our previous studies have shown increased generation of Ins(1,4,5)P₃ in atrial tissue from patients with valve disease, in hypertrophied ventricle from mice following chronic pressure overload, in atria and ventricles of the murine DCM model and in rodent hearts subjected to acute ischemia/reperfusion ^{4, 8, 26–28}. Other laboratories have also reported heightened Ins(1,4,5)P₃ generation in hearts undergoing hypertrophy ²⁹ or following ischemia or ischemia/reperfusion ^30–32. Thus, heightened generation of Ins(1,4,5)P₃, like increased IP₃-R expression, may be a common feature in cardiac pathologies. It is important to note that production of Ins(1,4,5)P₃ is an indication of phospholipase C activation following receptor activation. Ins(1,4,5)P₃ is generated from the sarcolemmal phospholipid phosphatidylinositol(4,5)bisphosphate (PIP₂) along with sn-1,2-diacylglycerol (DAG). Thus, increases in Ins(1,4,5)P₃ indicate increased DAG generation to activate subtypes of protein kinase C and protein kinase D ^{33, 34}, as well as some sarcolemmal canonical transient receptor potential (TrpC) channels ³⁵. Any of these DAG-induced responses could have major influences on disease progression that are independent of IP₃-R. Changes in the precursor PIP₂ also may be of critical significance, as this lipid is a direct regulator of the activity of a range of sarcolemmal ion channels critical in the maintenance of cardiac rhythm^35–38. It is also possible that Ins(1,4,5)P₃ has functions other than those that require IP₃-R activation. Ins(1,4,5)P₃ is the precursor of the highly phosphorylated inositol derivatives (InsP4-InsP8) ^{39, 40}, some of which are known to be functionally important ⁴¹, but currently such functions have not been described in heart.

These studies show that IP₃-R(2) do not make a significant contribution to disease in the DCM-2Tg or TAC models despite the increased activity of the Ins(1,4,5)P₃-IP₃-R(2) axis. We have recently reported that IP₃-R(2) contribute to the regulation of pacemaker function in mice, by demonstrating a lowering of the Ca²⁺ responses (by approximately 12%) in isolated sinoatrial node tissue in IP₃-R(2) −/− mice ²¹. In the current study we did not detect any effect of deletion of IP₃-R(2) on heart rate in DCM-2Tg mice, TAC mice or in WT or sham operated littermates. However, this contribution of IP₃-R(2) to pacemaker activity would be expected to be buffered by the autonomic nervous system in in vivo studies.

In conclusion we have demonstrated that Ins(1,4,5)P₃ and its receptors do not contribute to disease in a murine models of DCM and of hypertrophy, both of which have heightened Ins(1,4,5)P₃ generation and increased IP₃-R(2) expression. This finding shows that heightened expression of IP₃-R in heart disease cannot be assumed to have functional significance.