Genetic background influences expression and function of the cation channel TRPM4 in the mouse heart

Transient receptor potential melastatin 4 (TRPM4) cation channels act in cardiomyocytes as a negative modulator of the L-type Ca2+ current. Ubiquitous Trpm4 deletion in mice leads to an increased β-adrenergic inotropy in healthy mice as well as after myocardial infarction. In this study, we set out to investigate cardiac inotropy in mice with cardiomyocyte-specific Trpm4 deletion. The results guided us to investigate the relevance of TRPM4 for catecholamine-evoked Ca2+ signaling in cardiomyocytes and inotropy in vivo in TRPM4-deficient mouse models of different genetic background. Cardiac hemodynamics were investigated using pressure–volume analysis. Surprisingly, an increased β-adrenergic inotropy was observed in global TRPM4-deficient mice on a 129SvJ genetic background, but the inotropic response was unaltered in mice with global and cardiomyocyte-specific TRPM4 deletion on the C57Bl/6N background. We found that the expression of TRPM4 proteins is about 78 ± 10% higher in wild-type mice on the 129SvJ versus C57Bl/6N background. In accordance with contractility measurements, our analysis of the intracellular Ca2+ transients revealed an increase in ISO-evoked Ca2+ rise in Trpm4-deficient cardiomyocytes of the 129SvJ strain, but not of the C57Bl/6N strain. No significant differences were observed between the two mouse strains in the expression of other regulators of cardiomyocyte Ca2+ homeostasis. We conclude that the relevance of TRPM4 for cardiac contractility depends on homeostatic TRPM4 expression levels or the genetic endowment in different mouse strains as well as on the health/disease status. Therefore, the concept of inhibiting TRPM4 channels to improve cardiac contractility needs to be carefully explored in specific strains and species and prospectively in different genetically diverse populations of patients.


Introduction
During cardiac contraction cardiomyocytes of the working myocardium undergo electro-mechanical coupling. With extracellular electrical excitation, voltage-gated L-Type Ca 2+ channels in the membrane of the transverse tubules opens followed by a Ca 2+ influx, which elevates [Ca 2+ ] i and thereby activates calcium-induced calcium release (CICR) initiating myocardial contraction [4,8]. Cardiac contractility is influenced by numerous regulatory mechanisms, including the adjustment by the sympathetic nervous system. β-adrenergic stimulation by catecholamines activates the adenylyl cyclase (AC) which converts ATP to cAMP. An elevation in cytosolic cAMP level in cardiomyocytes leads to the activation of cAMP-dependent protein kinase A (PKA), which increases the activity of various effector proteins [20,32]. PKA phosphorylation of the voltage-gated L-type Ca 2+ channel, the ryanodine receptor RyR2 and Phospholamban, which relieves the inhibition of the sarcoplasmic reticulum (SR) Ca 2+ ATPase SERCA2a, increases the intracellular Ca 2+ influx during systole and provide more rapid reuptake 1 3 70 Page 2 of 16 of Ca 2+ ions into the sarcoplasmic reticulum during diastole [4,15]. Consequently, in the following systole, a larger Ca 2+ transient is generated resulting in a higher contraction force [29].
Expression of the calcium-activated monovalent cation channel Transient Receptor Potential Melastatin 4 (TRPM4) has been found in the heart of humans as well as in the atrium and ventricle of the mouse heart. It is expressed in cardiomyocytes, cardiac conduction system as well as in endothelial cells and smooth muscle cells [5,11,16,17,22,26]. The gain of function mutations of the Trpm4 gene are strongly associated with familial arrhythmia syndromes such as Brugada syndrome and the progressive familial heart block type I [16,18,34]. Insights from Trpm4-deficient cardiomyocytes have identified TRPM4 as a negative modulator of the L-type Ca 2+ influx under β-adrenergic stimulation, and its deletion leads to an increased action potential duration and thereby results in an enhanced driving force for Ca 2+ through the L-Type calcium channel. Consequently, systolic Ca 2+ transients are elevated under β-adrenergic stimulation, and contractility of isolated left ventricular papillary muscles showed that β-adrenergic stimulation leads to an increased contractile force in TRPM4-deficient mice compared to wildtype controls [21,37]. Furthermore, hemodynamic measurements under increasing β-adrenergic stimulation with isoproterenol resulted in a stronger left ventricular inotropic response in TRPM4-deficient mice compared to controls. However, under basal conditions, no significant differences were reported [21]. In addition, an enhanced β-adrenergic cardiac reserve was observed in TRPM4-deficient mice under conditions of ischemia evoked heart failure compared to wildtype controls [10]. These studies suggest a pharmacological or genetic inactivation of TRPM4 as a potential treatment strategy to enhance cardiac output in patients with severe heart failure with elevated catecholamine levels.
The identification of the genotype of the experimental mice was carried out by PCR using the primer pairs listed in Table 1. Ear biopsies were transferred into 97.5 μl of Direct-PCR buffer (Viagen, USA) with 2.5 μl proteinase K (10 mg/ ml, AppliChem, Germany) and lysed overnight at 55 °C. Subsequently, the proteinase K was denatured for 45 min at 85 °C.

Cardiac hemodynamic measurements
Mouse ventricular function was measured by a miniaturized pressure-conductance catheter. Mice were anesthetized by peritoneal injection of an anesthetic-analgesic mixture of 1200 mg/kg Urethane (Sigma-Aldrich, USA), 50 mg/ kg α-Chloralose (Sigma-Aldrich, USA), and 0. Optimal placement of the catheter was followed by a stabilization phase of 10 min. Preload dependent parameters were recorded by an expiratory ventilation break of max. 5 s. To assess preload independent contractility, the inferior caval vein was occluded and five consecutive PV loops were recorded during preload reduction to analyze Preload-Recruitable Stroke Work (PRSW). After basal recordings followed the β-adrenergic stimulation by infusion of increasing concentrations of the positive inotropic substance isoproterenol (9, 30, (60), 90 to a maximum of 300 ng/kg/min) [21,27]. At the end of each measurement, the parallel conductance of the myocardium was determined by an intravenous bolus injection of 15 μl of a 15% hypertonic NaCl solution. Cuvette calibration of heparinized blood from each mouse was used for conductance (µS) to blood volume (µl) conversion.

Statistics
Statistical analyses were performed using OriginPro 8.5 and Excel 2010. Data are expressed as mean ± standard error of the mean (SEM) or mean ± standard deviation (SD). Normality distribution of the data was tested using a Shapiro-Wilk test and considered normally distributed if p > 0.05. The difference between the two groups was tested using a Two-Sample T-test or its non-parametric alternative, Mann-Whitney test. P-values < 0.05 were considered statistically significant.

Results
Previous data from our group indicate that TRPM4 global knockout mice have an improved cardiac inotropic response to β-adrenergic stimulation [21]. To test whether the deletion of the Trpm4 gene specifically in cardiomyocytes improves cardiac contractility in healthy mice, we generated mice with a αMHC-Cre dependent deletion of the Trpm4 gene.
To analyze the efficiency of cardiomyocyte-specific Trpm4 deletion in Trpm4 flox/flox ; αMHC-Cre pos (Trpm4 iCM−KO ) mice, we isolated microsomal membrane fractions from hearts of three independent Trpm4 flox/flox ; αMHC-Cre pos and Trpm4 flox/flox ; αMHC-Cre neg mice, respectively, and performed Western Blot analysis. Proteins of ∼ 136 kDa were detected in the heart of Trpm4 flox/flox ; αMHC-Cre neg using ab478, which identified TRPM4 specifically in other cells and tissues from WT but not from the corresponding Trpm4 -/controls [22]. The size of these proteins is within the expected size range for TRPM4 proteins and the expression level of these proteins was reduced by 78 ± 10% in Trpm4 iCM−KO mice. Trpm4 -/mice entirely lacking TRPM4 proteins served as control (Fig. 1a). Using these mice, cardiac function was assessed using Pressure-Volume loop (PV-loop) measurements. After recordings of basal heart function, acute β-adrenergic stimulation with isoproterenol in Trpm4 iCM−KO and Cre neg control mice was performed ( Fig. 2a and b). A gradual increase in isoproterenol concentrations from 9 to 300 ng/kg/min resulted in the expected rise in heart rate ( Fig. 2c) and systolic parameters such as stroke volume (Fig. 2d) stroke work (Fig. 2e) and the contractility parameter Pre-recruitable Stroke Work (PRSW) (Fig. 2f). Additional parameters describing cardiac function were analyzed and are listed in Table 2. However, the results of the functional measurements showed no significant difference in cardiac contractile function between Cre pos and Cre neg animals.
Since all cardiac functional parameters analyzed were unaltered in Trpm4 -/-(Bl/6N) mice compared to corresponding Trpm4 +/+(Bl/6N) controls, we wanted to determine whether the differences to previously published results showing increased contractility in Trpm4 -/mice on a 129SvJ background upon isoproterenol treatment in PV loop measurements [21] and isolated papillary muscle preparations [37], were due to the different genetic background. Thus, we evaluated Trpm4 -/-(129SvJ) mice in comparison to litter matched Trpm4 +/+(129SvJ) controls. Western Blot analysis in this mouse line showed an abundant expression of ∼ 136 kDa proteins in Trpm4 +/+(129SvJ) mice, whereas these proteins were absent in Trpm4 -/-(129SvJ) mice (Fig. 1b, c). Again, measurements of basal heart function were followed by acute β-adrenergic stimulation via infusion of increasing isoproterenol concentrations from 9 to 300 ng/kg/min, which resulted in expected dose-dependent changes in PV loops in TRPM4 −/−(129SvJ) mice as well as in TRPM4 +/+(129SvJ) littermates ( Fig. 4a and f). The evaluation of the functional parameters showed no change in heart rate between TRPM4 −/−(129SvJ) and TRPM4 +/+(129SvJ) littermates (Fig. 4c). In contrast, a significantly increased β-adrenergic inotropic response in stroke volume (Fig. 4d) and in the left ventricular contractility parameter PRSW (Fig. 4f), which represents a key parameter to characterize cardiac function independent of preload and afterload [1,9], was found in TRPM4 −/−(129SvJ) mice compared to TRPM4 +/+(129SvJ) control mice. Additional parameters describing cardiac function were analyzed and are listed in Table 4.
Since differences in Ca 2+ signaling and contractility have been reported between C57Bl/6N and 129SvJ mouse strains before, which were attributed to differences in the expression of regulators of cardiomyocyte Ca 2+ homeostasis such as voltage-gated L-type channels, the SERCA2a ATPase or others [41] we tested whether the  Table 3 Hemodynamic analysis of constitutively TRPM4-deficient mice on the genetic C57Bl/6 N background. TRPM4 +/+ , n = 8; TRPM4 −/− , n = 7 HR, heart rate; SV, stroke volume; CO, cardiac output; Ved, end-diastolic volume; Ves, end-systolic volume; Ped, end-diastolic pressure; SW, stroke work; dP/dt max , peak rate of pressure rise; -dP/dt min , peak rate of pressure decline; PRSW, preload recruited stroke work; Tau  expression of SERCA2a and of Ca v β 2 , the predominant accessory subunit of cardiac L-type channels [23,42], might be differently expressed between cardiomyocytes from the two mouse strains. However, the densitometric analysis revealed no significant differences in these proteins between the two mouse strains (Fig. 1d, e). Notably, we observed significant differences in the abundance of TRPM4 proteins, which were 78 ± 10% higher in wildtype mice on the 129SvJ genetic background compared to the C57Bl/6N strain (Fig. 1c). Furthermore, we found no difference in the caffeine-evoked Ca 2+ amplitude between cardiomyocytes of the 129SvJ strain and the C57Bl/6N strain indicating that the amount of Ca 2+ which can be released from the SR is not different in cardiomyocytes of these two mouse strains (Fig. 5e, j).

Discussion
Our previous studies have suggested that the activation of the cation channel TRPM4 in cardiomyocytes reduces the driving force for L-type mediated Ca 2+ influx. The global deletion of Trpm4 in the mouse model resulted in increased cardiac contractility under β-adrenergic stimulation, both under healthy conditions [21,37] and after induction of myocardial infarction [12]. Our current results suggest that in healthy  Table 4 Hemodynamic analysis of constitutively TRPM4-deficient mice on the genetic 129SvJ background. TRPM4 +/+ , n = 7; TRPM4 −/− , n = 6 Significant differences between TRM4 +/+ and TRPM4 -/-are indicated as bold values HR, heart rate; SV, stroke volume; CO, cardiac output; Ved, end-diastolic volume; Ves, end-systolic volume; Ped, end-diastolic pressure; SW, stroke work; dP/dt max , peak rate of pressure rise; -dP/dt min , peak rate of pressure decline; PRSW, preload recruited stroke work; Tau  mice this effect is critically dependent on the genetic background of the mouse model being studied. Indeed, both in an inducible cardiomyocyte-specific Trpm4 flox/flox ; αMHC-Cre ERT2 mouse model as well as in globally TRPM4-deficient mice on the same C57Bl/6N genetic background no increase in inotropy during β-adrenergic stimulation could be detected. Of note, a difference in the protocol used in this study is that the pressure-volume catheter is placed in the  [21]. The latter, previously published, PV loop measurements showed an increased ISO-evoked improvement of contractility in Trpm4 -/mice on the 129SvJ background [21], which we could faithfully reproduce in this study, in global Trpm4 -/-(129SvJ) mice in comparison to Trpm4 +/+(129SvJ) littermates using open-chest insertion of the catheter via the apex. Determination of left ventricular function revealed a significantly increased β-adrenergic inotropy, as well as a significant increase in stroke volume during isoproterenol stimulation in Trpm4 -/-(129SvJ) mice in comparison to Trpm4 +/+(129SvJ ), controls. This indicates that the difference in the methodological approach cannot explain why an increase in ISO-evoked cardiac contractility was not observed upon TRPM4 deletion in healthy mice of the C57Bl/6N strain in our study. Since this phenomenon was confined to Trpm4 -/-(129SvJ) mice, we conclude that the phenotype of increased β-adrenergic inotropy depends on the genetic background, as well as on the health/disease status of the mice. The influence of genetic background on cardiac functions has been demonstrated in several other studies. The genetic homogeneity of inbred strains provides ideal conditions for the investigation of causal physiological relationships. The most commonly used genetic background in the analysis of cardiac function is that of the C57Bl/6 mouse, the first mouse strain that was fully sequenced [24]. Most genetically modified mouse lines were generated from embryonic stem cells from inbred strains of the 129 background and then backcrossed to the genetic background of C57Bl/6 mouse strains. However, there are genetic divergences between different inbred strains, which are often paid little attention and can lead to confusing results when comparing different inbred strains [6]. For example, C57Bl/6 mouse strains have a single renin gene, Ren-1c, while 129SvJ mice have two renin genes, Ren-1d and Ren-2. This is associated with e.g. higher blood pressure in 129SvJ compared to C57Bl/6 mice [19]. Further studies reported differences in cardiac function between different inbred strains [2,3,27,30,31,35,38,41]. Hemodynamic analysis of eight commonly used inbred mouse strains showed differences in cardiac contractility that were pronounced during acute hypoxia or β-adrenergic blockade [2]. The β-adrenergic blockade inhibited cardiac function more strongly in 129X1/SvJ than in the C57Bl/6J strain suggesting that 129X1/SvJ mice have a higher sympathetic basal tone than C57Bl/6J mice, which in addition would explain desensitization-induced lower cardiac β1 adrenoceptor density in 129X1/SvJ animals compared to C57Bl/6J mice [31]. Differences in contractility and relaxation between the 129SvJ and C57Bl/6 J strain with and without ISO treatment were reported using hemodynamic measurements [41]. Similar studies with comparative analysis of the cardiac contractile function of the C57Bl/6N strain have not been reported to our knowledge. The distinction between N and J strain is of importance for the analysis of cardiac function particularly in the disease state. A recent study could demonstrate in detail that the defective mitochondrial transhydrogenase (NnT) in C57Bl/6J ameliorates pressure overload-induced cardiac failure because of differences in antioxidative capacity [25].
Interestingly, differences in the expression of proteins affecting the intracellular Ca 2+ transient were also observed between different inbred strains. Quantitative PCR analysis showed a significantly higher expression of RyR2 and an enhanced expression of SERCA2a in 129/SvJ mice compared to C57Bl/6 [41]. This could potentially affect intracellular Ca 2+ homeostasis, as Shah and colleagues had pointed out. They recognized that cardiomyocytes from 129/SvJ mice have a higher sarcoplasmic reticulum (SR) "Ca 2+ -load" and a lower SR "Ca 2+ -leak" compared to C57Bl/6 mice [31]. However, under a frequency-dependent stimulation, no increase in systolic Ca 2+ amplitude could be detected between the cardiomyocytes of the two strains, as well as no difference in SR Ca 2+ release [31]. Also, in our hands, no obvious differences in the Ca 2+ handling between 129SvJ and C57Bl/6N cardiomyocytes could be observed. In contrast, we found that the expression of TRPM4 proteins is about 78 ± 10% higher in wild-type mice on the 129SvJ genetic background compared to the C57Bl/6N strain. This interesting finding could indicate that the TRPM4 protein is more physiologically relevant, particularly with respect to inotropic response, in the left ventricle of 129SvJ mice than in C57Bl/6N mice. Mathar et al. suggested that the inotropic phenotype of TRPM4 −/− mice depends essentially on a modification of Ca 2+ dependent excitation-contraction coupling in TRPM4 −/− mice. They found that the ISOinduced increase of the evoked Ca 2+ transient is significantly larger in TRPM4 −/− cardiomyocytes, compared to WT cardiomyocytes. Considering that differences in Ca 2+ signaling in cardiomyocytes from different mouse strains have been shown before [31], we studied catecholamine evoked rise in systolic Ca 2+ transients in cardiomyocytes of both genotypes and mouse strains. Whereas the ISO-evoked increase of the Ca 2+ transient was significantly larger in TRPM4 −/− cardiomyocytes in the 129SvJ strain compared to TRPM4 +/+ controls, which reinforces our previous analysis [21], we could not find a significant difference between the two genotypes in the C57Bl/6 N background. In our attempts to gain mechanistic insights into the differences in TRPM4dependent Ca 2+ signaling between the two strains, we analysed protein expression of the L-Type Ca 2+ channel subunit Ca V β 2 and SERCA2a proteins, whose expression level and activity regulate diastolic SR Ca 2+ load [7]. However, our findings show neither divergences in Ca V β 2 and SERCA2a protein expression nor differences in caffeine-induced SR Ca 2+ depletion in cardiomyocytes from 129SvJ compared to C57Bl/6N mice indicating that SR Ca 2+ load is not different in ventricular cardiomyocytes between the strains. Taken together, these results confirm our previous analysis. The finding that cardiac TRPM4 expression in C57Bl/6N was drastically (∼ 80%) lower compared 129SvJ mice suggests that TRPM4 is a relatively less important determinant of the driving force of L-type Ca 2+ entry in C57Bl/6N cardiomyocytes, as compared to 129SvJ cardiomyocytes.
However, we cannot exclude that also other factors that determine ISO-evoked Ca 2+ rise and cardiac contractility on the organismal level might be different between TRPM4deficient mice on the respective genetic background. An example of such discrepancies was reported in the study of Vignier et al. who investigated the LMNA p.H222P mutation leading to Emery-Dreifuss muscular dystrophy (EDMD) in humans in C57BL/6JRj and 129S2/SvPas background. This mutation is associated with arrhythmia-related symptoms resulting in cardiac dilation and heart failure. In contrast to C57 Lmna p.H222P mice, 129 Lmna p.H222P mice showed exacerbated arrhythmia susceptibility making 129 Lmna p.H222P mice a more relevant model for evaluating therapeutic strategies for EDMD [40]. The studies above as well as our findings emphasize that the analysis of null alleles or disease-related mutations in multiple mouse strains is recommendable. Ultimately, the most adequate species and strain need to be chosen to study distinct physiological or pathophysiological processes [2,13,38,41] and to test potential therapeutic strategies. Obviously, the 129SvJ strain is more suitable to study TRPM4-dependent processes in the heart compared to the C57Bl/6N strain where the homeostatic expression level is comparably low.
Overall, it can be concluded that a genotype-phenotype context of an isolated genetic background is not easily generalized for different inbred mouse strains [28,33]. F1 hybrids could be a useful alternative to investigate the influence of TRPM4 deficiency in a more complex genetic background. In a previous study by Uhl et al., TRPM4-deficient 129B6F1 hybrids were used, which under β-adrenergic stimulation with isoproterenol achieved an increased positive inotropic response in isolated left ventricular papillary muscles compared to wild-type 129B6F1 hybrids [37]. However, it remains to be investigated whether cardiac contractility in the TRPM4-deficient 129B6F1 hybrid mouse also leads to an increased β-adrenergic positive inotropy in vivo. The extent of relevance of TRPM4 deletion may also be differently pronounced between healthy mice and different degrees of disease status. Whereas in this study the inotropic response was unaltered in Trpm4 -/-(Bl/6N) mice of the C57Bl/6N background, it could be shown that the β-adrenergic reserve was preserved in Trpm4 -/-(Bl/6N) mice after permanent LAD ligation and significantly higher compared to Trpm4 +/+(Bl/6N) leading to an increased survival rate [12]. A potential beneficial effect of inactivating or deleting TRPM4 in other cardiac disease models in the mouse or other species has not been investigated so far.
From a translational point of view, a single isogenic mouse strain is limited in its genetic diversity and may not reflect the physiological responses of the human polymorphic population [14]. The analysis of a null mutation in multiple mouse strains offers the opportunity to increase the genetic diversity of the mouse model, which allows comparison with the broad spectrum of human genetic heterogeneity [28]. Thus, the concept of inhibiting TRPM4 channels or suppressing their expression needs to be explored in different genetically diverse populations of patients suffering from cardiac failure.
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