Rapid Multi-Slice T₁ Mapping of Mouse Myocardium: Application to Quantification of Manganese Uptake in α-Dystrobrevin Knockout Mice

Abstract

Purpose

The aim of this study was to develop a rapid, multi-slice cardiac T₁ mapping method in mice, and to apply the method to quantify manganese (Mn²⁺) uptake in a mouse model with altered Ca²⁺ channel activity.

Methods

An ECG-triggered multi-slice saturation-recovery Look-Locker method was developed and validated both in vitro and in vivo. A two-dose study was performed to investigate the kinetics of T₁ shortening, Mn²⁺ relaxivity in myocardium, and the impact of Mn²⁺ on cardiac function. The sensitivity of Mn²⁺-enhanced MRI in detecting subtle changes in altered Ca²⁺ channel activity was evaluated in a mouse model with α-dystrobrevin knockout.

Results

Validation studies showed strong agreement between the current method and an established method. High Mn²⁺ dose led to significantly accelerated T₁ shortening. Heart rate decreased during Mn²⁺ infusion, while ejection ratio increased slightly at the end of imaging protocol. No statistical difference in cardiac function was detected between the two dose groups. Mice with α-dystrobrevin-knockout showed enhanced Mn²⁺ uptake in vivo. In vitro patch-clamp study showed increased Ca²⁺ channel activity.

Conclusion

The saturation-recovery method provides rapid T₁ mapping in mouse hearts, which allowed sensitive detection of subtle changes in Mn²⁺ uptake in α-dystrobrevin-knockout mice.

INTRODUCTION

Calcium (Ca²⁺) is the central regulator in cardiac excitation-contraction coupling (1). Ca²⁺ influx via the voltage-gated L-type Ca²⁺ channels, which triggers Ca²⁺-induced Ca²⁺ release from sarcoplasmic reticulum, is of key importance in cardiac Ca²⁺ cycling. Despite the importance of Ca²⁺ uptake, limited imaging techniques exist for noninvasive measurement of Ca²⁺ channel activity. The emergence of manganese (Mn²⁺)-enhanced magnetic resonance imaging (MEMRI) provides the possibility for in vivo assessment of Ca²⁺ uptake (2). Because of its T₁ shortening effect and its long retention in cells, Mn²⁺ has become a useful molecular contrast agent in cardiac MRI (3–5). Unlike gadolinium-based contrast agents which are trapped in the extracellular space (6–8), Mn²⁺ enters the cell via the Ca²⁺ channels (9–12). Hence, the accumulation of Mn²⁺ in myocardium reflects Ca²⁺ channel activity and thus Ca²⁺ influx. As such, MEMRI offers a novel means for measuring Ca²⁺ channel activity in hearts.

T₁-weighting and T₁ mapping are the two widely used methods to characterize Mn²⁺ uptake in MEMRI (10,13–16). The T₁-weighting method offers high temporal resolution. However, it does not provide direct quantification of Mn²⁺ concentration in the cells. The T₁ mapping method, in contrast, offers quantitative measurement of the Mn²⁺ accumulation because of the linear relationship between the relaxation rate (R₁=1/T₁) and Mn²⁺ concentration within a certain range (4,13,14,17). To quantify the Ca²⁺ channel activity, T₁ mapping with high temporal resolution is required to measure the kinetics of Mn²⁺ uptake (14,15). Previously, a Saturation-Recovery Look-Locker (SRLL) method was developed and validated in our lab for fast cardiac T₁ mapping in mice (18). This method allowed acquisition of single slice T₁ maps of mouse myocardium within 3 min. Despite a significant improvement in temporal resolution, T₁ mapping of a single slice by SRLL provides limited coverage of the whole heart.

Quantification of the concentration of Mn²⁺ within tissue from measured T₁ relaxation times requires knowledge of the relaxivity (r₁) of Mn²⁺ within myocardium, where r₁ is the rate of R₁ increase when Mn²⁺ concentration increases. To date, only a few studies have attempted to quantify r₁ in myocardium in vivo (4,13,19). Further, the optimal dose for MEMRI studies is yet to be determined. While high Mn²⁺ dose provides greater sensitivity to detect changes in Ca²⁺ channel activity, a high tissue Mn²⁺ concentration may saturate the relaxation effect induced by Mn²⁺, leading to a nonlinear increase in R₁ and inaccurate quantification of Mn²⁺ concentration (10,13,19). In addition, high myocardial Mn²⁺ accumulation may negatively impact ventricular function (2,15,20,21). Thus, the optimal Mn²⁺ dose must balance sensitivity and accuracy in delineating Ca²⁺ channel activity while maintaining normal ventricular function.

In the current study, the previous SRLL method was modified to achieve multi-slice T₁ mapping (MSRLL) in mouse heart with no additional time cost. In vitro and in vivo studies were performed to validate the MSRLL method with the previously established SRLL method. An in vivo study with two doses of MnCl₂ solutions was performed to investigate the relationship between R₁ and Mn²⁺ concentrations in myocardium, as well as the impact of Mn²⁺ on cardiac function. The utility of MSRLL was further evaluated in a mouse model with α-dystrobrevin knockout. Our results show that the MSRLL method can provide rapid multi-slice T₁ mapping in mouse heart that allows sensitive detection of subtle changes in altered Ca²⁺ channel activity.

METHODS

Imaging Method

A schematic diagram of the MSRLL pulse sequence is shown in Fig. 1. An ECG-triggered saturation module was applied at the beginning of each phase-encoding step. It consisted of three nonselective 90° radio-frequency pulses to ensure complete saturation of the magnetization. The saturation module was followed by the acquisition of k-space lines of n×m images using fast low-angle shot (FLASH) method, where n is the number of images acquired along the magnetization recovery curve, and m is the total number of slices. The acquisition of the FLASH images was triggered by the detection of the QRS complex, with 80~100 ms delay after the trigger to ensure that all the images were acquired during mid to late diastole. Multi-slice images were acquired during the diastolic period using the interlaced scheme to minimize the crosstalk between adjacent slices.

Figure 1. MSRLL pulse sequence.

After the saturation module, 10 or 20 sequential ECG-triggered, multi-slice FLASH acquisitions separated by an interval τ were implemented. Each block on the relaxation curve represents a multi-slice acquisition module. To minimize cardiac motion artifact, a trigger delay was applied before the FLASH acquisitions such that all the images were acquired at mid-to-late diastole.

Phantom Study

All MRI studies were performed on a horizontal 7T Bruker scanner (Bruker Biospec, Germany) using a 35-mm inner diameter volume coil. The MSRLL method was first validated in vitro using a multi-compartment phantom with manganese chloride (MnCl₂) solutions sealed in 1 mL centrifuge tubes. The MnCl₂ concentrations ranged from 30 to 1000 μM. MSRLL was applied to acquire T₁ maps of 5 slices with 0.1 mm inter-slice gap. 1-ms Hermite pulses were used for slice excitation. The imaging parameters were: flip angle, 10°; TE, 1.9 ms; slice thickness, 1 mm; number of averages, 1; field of view, 3.0×3.0 cm²; matrix size 128×64. For each slice, a total of 10 images that covered 2.4 s of the saturation recovery curve were acquired with a 240 ms interval. For each image, a single k-space line for each of the 5 slices was acquired within 23.6 ms. Single slice SRLL images were also acquired to validate the MSRLL results. Proton density (M₀) images were acquired with a TR of 3 s and the same flip angle as the MSRLL acquisition.

Animals and In Vivo Studies

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Case Western Reserve University. 3 month, male FVB mice (n=19) were used for in vivo validation and the two-dose MEMRI studies. Specifically, mice in prone position were anesthetized with 2% isoflurane and maintained with 0.8–1.8% isoflurane. A 26G×19 mm plastic IV catheter (Hospira, Inc., Lake Forest, IL) was inserted into the tail vein and connected to an infusion pump (Braintree Scientific, Inc., Braintree, MA) for Mn²⁺ infusion. ECG, respiration, and body temperature were monitored and recorded by a physiological monitoring system (SA Instruments, Stony Brook, NY). Hot air was blown to the mice to maintain the body temperature at around 35°C.

To investigate the effect of different sampling intervals on T₁ fitting, two ECG triggering schemes were applied during the acquisition of FLASH images: triggering at every two heartbeats and triggering at every heartbeat, equivalent to a sampling interval of 2 R-R intervals and 1 R-R interval along the recovery curve, respectively. T₁ maps of three adjacent short-axis slices at mid-ventricular levels were acquired using MSRLL. To capture the magnetization recovery at an early phase, the first set of multi-slice images were acquired 40~80 ms after the implementation of the saturation module. All images were acquired during mid- to late-diastole to minimize motion artifact. Imaging parameters were: flip angle, 10°; TE, 1.9 ms; slice thickness, 1 mm; number of averages, 1; field of view, 3.0×3.0 cm²; matrix size, 128×64. During each phase-encoding step, a single k-space line for each of the three slices was acquired within 18.7 ms. For each slice, a total of 10 (triggering at every two heartbeats) or 20 images (triggering at every heartbeat) were acquired, covering approximately ~2.4 s of the saturation recovery curve. SRLL images were also acquired using the corresponding triggering scheme to validate the MSRLL results. Proton density images (M₀) were acquired with a TR of 10 R-R intervals (1100~1300 ms).

In vivo MEMRI experiments were conducted to demonstrate the utility of MSRLL in delineating dynamic changes in T₁ relaxation times. After the acquisition of baseline T₁ maps, MnCl₂ solution was infused through the tail vein catheter at a rate of 0.2 mL/hr for 30 minutes, followed by a 15 minutes washout period. Two different Mn²⁺ concentrations were used: 126 mM (n=9, referred to as the high dose group) and 63 mM (n=10, referred to as the low dose group). T₁ maps for the high dose group were acquired by triggering at every two heartbeats, while those for the low dose group were acquired by triggering at every heartbeat. T₁ maps were acquired continuously during Mn²⁺ infusion and washout. Heart rate was continuously recorded during the whole imaging protocol. Cine FLASH images of the mid-ventricular slices were acquired at baseline and post-contrast to evaluate the impact of Mn²⁺ on ventricular function. Validation study by SRLL was performed either before Mn²⁺ infusion (n=5 for each dose group) or after the 15-min washout period (n=5 for the high dose group and n=4 for the low dose group).

To further evaluate the sensitivity of MSRLL in detecting subtle changes in Ca²⁺ channel activity, the kinetics of Mn²⁺-induced R₁ increase was measured in 4 to 5 month old, male α-dystrobrevin knockout mice (adbn^−/−, n=5) and their age-matched controls (C57BL/6, n=5). The adbn^−/− mice have been described previously (22). Mn²⁺ infusion used the high dose and image acquisition was triggered at every heartbeat.

Image Analysis

Images were analyzed off-line using MATLAB-based software developed in-house (18). Images were zero-filled to 128×128 matrix during reconstruction. For each slice, T₁ maps of the whole left-ventricle were generated by performing pixel-wise curve fitting. The details on T₁ fitting have been described previously (18). Briefly, the modified relaxation time (T₁^*) and steady-state magnetization (M^*) maps were obtained from pixel-wise, least-square fitting of a three-parameter mono-exponential function to the acquired Look-Locker images. Subsequently, T₁ map was calculated from the T₁^*, M^*, and M₀ maps using the following equation:

$$T_1=\frac{M_0}{M^{\ast }}{T_1}^{\ast }$$

For the analysis of CINE images, epicardial and endocardial contours were traced manually. The ejection ratio was calculated as the percentage change in the area that encompassed the left-ventricle from end-diastole to peak systole.

Quantification of Mn²⁺ Concentration and Relaxivity in Myocardium

Hearts from both dose groups were excised and freeze-clamped after imaging studies. The frozen tissues were burned in a furnace (Thermodyne, Salt Lake City, UT) at 600°C for 3 hours. The ashes were dissolved in 20% nitric acid and the Mn²⁺ content was analyzed by a flame atomic absorption spectrophotometer (Buck Scientific, East Norwalk, CT) as previously described (15). Regression analysis was performed to quantify the Mn²⁺ relaxivity in myocardium. Mn²⁺ content at baseline was considered negligible.

Myocyte Isolation

Ventricular myocytes were isolated using the method described by Ren et al with minor modifications (23). Briefly, mice were injected with heparin (1000 units/kg, i.p.) and sacrificed by cervical dislocation. Hearts were quickly excised, cannulated, and perfused with Ca²⁺-free Tyrode solution. The Tyrode solution contained (in mM) 136 NaCl, 5.4 KCl, 1.0 MgSO₄, 1.2 NaH₂PO₄, 10 HEPES, 5.6 glucose, 2 L-glutamine, 5 taurine, and 0.03 L-ascorbic acid, 1X vitamins and amino acids (Gibco, Invitrogen Corporation). The pH of the solution was adjusted to 7.4 with NaOH. Afterwards, hearts were perfused with Tyrode solution containing 0.8 mg/mL collagenase type II (Worthington Biochemical Co., Lakewood, NJ, USA) for 8~10 min. The ventricles were removed from the perfusion column, minced, gently agitated and rinsed. Isolated myocytes were collected and stored in Media 199 (GIBCO, Grand Island, NY, USA) containing 1.8 mM Ca²⁺. All cardiomyocytes were used within 3 hours after isolation. Only rod-shaped myocytes with clear striations were selected.

L-type Calcium Current (I_Ca) Measurement

Calcium currents were recorded in isolated ventricular myocytes using conventional whole cell patch clamp techniques. Data acquisition was performed using pClamp 9 software and an Axopatch 200A patch clamp amplifier (Molecular Devices). The pipette solution contained (in mM) 125 CsCl, 20 tetraethylammonium chloride, 5 MgATP, 3.6 creatine phosphate, 10 EGTA, 10 HEPES, pH 7.2. The bath solution contained (in mM) 157 tetraethylammonium chloride, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, pH 7.4. Patch pipettes had a resistance of 2–4 MΩ. Series resistance compensation (50–80%) was used to minimize voltage control errors. Ca²⁺ current recordings began 3 min after patch rupture. Ca²⁺ currents were normalized to cell capacitance (pA/pF) to account for variations in cell size. Electrophysiologic experiments were performed under continuous flow conditions at 36.5±0.5°C.

Statistical Analysis

All results were expressed as mean ± standard deviation (SD). Comparison of T₁ values by MSRLL and SRLL was performed by paired student’s t-test and Bland-Altman analysis. Paired student’s t-test was also used to compare the heart rate and ejection ratio during Mn²⁺ infusion and washout with that at the baseline. Unpaired Student’s t-test was used for comparison between two groups. Two-way repeated measures ANOVA was performed to compare the time courses of R₁ increase and the time courses of heart rate changes during the imaging protocol. P values less than 0.05 were considered statistically significant.

RESULTS

Phantom Study

Total acquisition time was 2 min and 40 s for phantom studies. Shown in Fig. 2a are the T₁ maps of all five slices. There were no statistical differences in measured T₁ values among all five slices. Regression analysis showed a linear relationship between Mn²⁺ concentration and the mean R₁ for each of the five slices (Fig. 2b). There was a strong agreement between T₁ measurements by MSRLL and SRLL methods (Fig. 2b). The in vitro relaxivity of Mn²⁺ measured by MSRLL and SRLL was also similar (5.01 versus 5.04 mM⁻¹s⁻¹).

Figure 2. T₁ mapping of the multi-compartment phantom.

a. T₁ maps of the five imaging slices. Phantoms 1–8 contain MnCl₂ solutions with the following concentrations (μM): 1000, 900, 700, 500, 300, 200, 100, and 30. b. Quantitative comparison of the T₁ measurements by MSRLL and SRLL. c. Bland-Altman plot of the difference between T₁ measured by MSRLL and SRLL. The middle dotted line is the mean of the difference. The upper and bottom dotted lines are the mean plus and minus two standard deviations, respectively.

Pixel-by-pixel Bland-Altman analysis of the difference between MSRLL and SRLL measurements is shown in Fig. 2c. With short T₁ (<1 s), there was a strong agreement between MSRLL and SRLL measurements. The trend of increased difference with longer T₁ was due to insufficient coverage of the saturation recovery curves as was demonstrated by previous simulation studies (18).

In Vivo Validation Study

Average acquisition time for in vivo studies was 140~166 s (R-R interval: 110~130 ms). Representative T₁ maps from the low dose groups by MSRLL and SRLL are shown in Fig. 3a. Quantitative comparison showed no difference in T₁ measurements between SRLL and MSRLL methods both at baseline and after Mn²⁺ washout (Fig. 3b). Pixel-by-pixel Bland-Altman analysis also showed strong agreement in T₁ values measured by MSRLL and SRLL for both triggering schemes (Fig. 4), especially in post-contrast T₁ mapping. Compared with triggering at every two heartbeats (Fig. 4a), triggering at every heartbeat (Fig. 4b) showed more consistent agreement between MSRLL and SRLL measurements. In addition, triggering at every heartbeat yielded better separation between the pre- and post-contrast T₁ values. There were also fewer data points with the difference between the two measurements larger than 2 standard deviations.

Figure 3. In vivo T₁ mapping.

a. Representative T₁ maps at pre- (left) and post-contrast (right) acquired by MSRLL (top) and SRLL (bottom), respectively. b. Comparison of T₁ mapping by MSRLL and SRLL.

Figure 4. Bland-Altman plots for the comparison of T₁ measurements by MSRLL and SRLL in vivo.

a. High Mn²⁺ dose group. b. Low Mn²⁺ dose group. The middle dotted line is the mean of the difference. The upper and bottom dotted lines are the mean plus and minus two standard deviations, respectively.

Representative T₁ maps acquired at baseline and after Mn²⁺ infusion are shown in Fig. 5a&b. The time courses of the R₁ changes for all three slices are presented in Fig. 5c. For each dose group, there were no statistical differences in measured T₁ among all three slices. ANOVA analysis showed significantly larger increase in R₁ for all three slices in the high dose group (P<0.05). In the two slices that are distal to the apex, the high dose group also showed significantly larger increase of R₁ from 18 to 27 min during Mn²⁺ infusion (P<0.05). The apical slice showed a larger standard deviation in R₁. As a result, statistical significance was only detected at 18 min of Mn²⁺ infusion. R₁ values during the washout period were similar between the two dose groups.

Figure 5. R₁ changes in dynamic MEMRI study.

a&b. Pre- and post-contrast T₁ maps of the apical (left), mid-ventricular (middle), and basal slices (right), respectively. c. Time courses of R₁ changes. *P<0.05 high dose versus low dose, with the color of * indicating the corresponding slice.

The impact of Mn²⁺ accumulation on heart rate and ejection ratio is shown in Fig. 6. Comparing to the baseline, a 6.1% and 4.2% decrease in heart rate was observed in the high and low dose groups, respectively (Fig. 6a). ANOVA analysis detected no difference in the time courses of heart rate between the two dose groups. Fig. 6b shows the ejection ratio of the mid-ventricular slice at baseline and post-contrast. Comparing to the baseline, there was a slight increase in ejection ratio in both dose groups (P<0.05).

Figure 6. The impact of Mn²⁺ accumulation on heart rate and ejection ratio.

a. Time courses of the heart rate at low and high Mn²⁺ doses. b. Ejection ratio of the mid-ventricular slice at pre- and post-contrast. *P<0.05 compared to heart rate at pre-contrast in the high dose group; ^†P<0.05 compared to heart rate at baseline in the low dose group; ^#P<0.05 compared to ejection ratio at pre-contrast.

Mn²⁺ Concentration and Relaxivity in Myocardium

Mn²⁺ accumulation in myocardium was significantly higher in the high dose group (P<0.05, Fig. 7a). A progressive increase in R₁ was observed when the Mn²⁺ concentration was low. However, this increase reached a plateau at higher Mn²⁺ concentration (Fig. 7b). Regression analysis using data only from the low dose group yielded an r₁ of 6.02 mM⁻¹s⁻¹ with an R² of 0.87. When using data from both dose groups, the fitted r₁ was only 2.97 mM⁻¹s⁻¹ with a reduced R² of 0.53.

Figure 7. Mn²⁺ concentration and relaxivity in myocardium.

a. Post-contrast Mn²⁺ concentration in myocardium by flame atomic absorption spectrophotometry. *P<0.05 high dose versus low dose. b. Mn²⁺ relaxivity in myocardium by linear regression analysis. The solid and dashed lines represent the regression analysis using data from the low dose group and both dose groups, respectively.

In Vivo MEMRI on adbn^−/− Mice

Shown in Fig. 8a are the time courses of the R₁ changes for all three slices in adbn^−/− mice and their controls. There was a significantly larger increase in R₁ in adbn^−/− mice for all three slices by ANOVA analysis (P<0.05). Unpaired Student’s t-test also showed significantly larger increase in R₁ from 3 to 18 min during Mn²⁺ infusion in adbn^−/− mice. Calculated Mn²⁺ concentration maps at 18 min during Mn²⁺ infusion for control and adbn^−/− mice are shown in Fig. 8b. Mean Mn²⁺ uptake calculated from R₁ changes is in agreement with that reported in literature (13). Unpaired Student’s t-test revealed a significantly higher Mn²⁺ uptake in adbn^−/− mice (Fig. 8c). No difference in R₁ was observed during late infusion (18~30 min) and washout (30~45 min).

Figure 8. Mn²⁺ uptake and Ca²⁺ channel activity in adbn^−/− mice.

a. Time courses of R₁ changes by MEMRI. b. Representative Mn²⁺ concentration maps at 18 min during Mn²⁺ infusion in adbn^−/− and control mice. c. Average Mn²⁺ concentration at 18 min of Mn²⁺ infusion. d. Capacitance normalized current-voltage relationship. e. Boltzman analysis of the potential for half-maximal activation (V_1/2) of the calcium current. *P<0.05 compared to control mice. The colors of * in panel a indicate the corresponding slice.

Alteration in Ca²⁺ Currents in adbn^−/− Mice

No difference was observed in cell capacitance between adbn^−/− and control myocytes (144±9 vs. 163±17 pF). Fig. 8d shows the capacitance normalized current-voltage relationship for I_Ca. While peak I_Ca density (at 0 mV) was not significantly different between the control and adbn^−/− mice, I_Ca density was greater in adbn^−/− myocytes at −10 and −20 mV (P<0.05). Boltzman analysis revealed a 4.5 mV hyperpolarizing shift in the potential for half-maximal activation (V_1/2) of I_Ca, from −19.8 mV in control myocytes to −24.3 mV in the adbn^−/− mice (Fig. 8e).

DISCUSSION

In the current study, we present a rapid, multi-slice T₁ mapping method that allows the measurement of T₁ in mouse myocardium at ~3 min temporal resolution. In vitro and in vivo validation studies demonstrated that the accuracy of our current method is comparable to that of the single-slice method developed and validated previously. The utility of this method in monitoring the dynamics of Mn²⁺-induced T₁ changes during Mn²⁺ infusion and subsequent washout was demonstrated in a two-dose in vivo MEMRI study. The sensitivity of rapid T₁ mapping in detecting subtle changes in Ca²⁺ channel activity was also evaluated in an MEMRI study comparing the adbn^−/− mice and their controls. In addition, Mn²⁺ relaxivity in myocardium, which allows the absolute quantification of tissue Mn²⁺ content from measured T₁, was estimated. Finally, Mn²⁺-induced changes in cardiac function were also evaluated.

The accuracy of T₁ estimation is dependent on the sampling of the T₁ recovery curve. In cardiac T₁ mapping, the sampling interval is determined by the ECG triggering scheme. To avoid mis-triggering caused by the switching gradients, a triggering scheme at every two heartbeats can be used, leading to a sampling interval of 2 R-R intervals, which may give rise to insufficient coverage of the ascending portion of the recovery curve, especially at shorter T₁ values. Alternatively, triggering at every heartbeat doubles the number of data points and improves the accuracy of T₁ fitting. To minimize mis-triggering, the acquisition of the FLASH images needs to be implemented at an earlier phase during diastole (mid- to late-diastole) such that the QRS complex of the ECG signal is less affected by the switching gradients. However, mis-triggering may still occur occasionally, especially when heart rate fluctuates. Further, since a mouse heart has no real period of diastasis, the slices acquired at mid to late diastole were at slightly different phases of diastole. The intra-slice T₁ variations observed in the current study may be partially attributed to measurement errors caused by mis-triggering or motion artifact, which may diminish the sensitivity of detecting true T₁ heterogeneity in a single T₁ map. However, as the current method is aimed at following the dynamics of Mn²⁺ uptake with multiple acquisitions of T₁ maps during Mn²⁺ infusion, such pixel-wise variations are likely to appear random over the time course of Mn²⁺ infusion.

T₁ estimation in the current study used a fixed data sampling interval that was calculated from the average heart rate during acquisition. Hence, variations in heart rate will lead to mis-assignment of the data points that can impact the accuracy of T₁ estimation. For example, a 10% change in heart rate will lead to a 10% error in T₁ estimation. In our current study, changes in heart rate (typically <10% and mostly around 5%) developed progressively during the ~3 min data acquisition. Since the acquisition of the center k-space lines, which reflects the majority of changes in signal intensity, occurred in even less time, the errors introduced by the mis-assignment of the data points should be much smaller than 10%.

While the imaging sequence was designed to perform multi-slice T₁ mapping that can cover the whole heart, T₁ mapping at the apical and basal slices will be less robust because myocardium at these two levels has more pronounced through-plane motion. In the current study, we imaged three adjacent 1 mm thick slices to demonstrate the utility of this method, which might be sufficient for most cardiac applications using the AHA 17 segment model. Further, previous studies have indicated the need for respiratory motion correction (24,25). However, simultaneous ECG and respiratory triggering will lead to non-uniform sampling intervals and prolonged data acquisition time. Since the focus of the current study is to develop a fast T₁ mapping method that can capture the kinetics of Mn²⁺ uptake in murine myocardium, no respiratory triggering was used to account for respiratory motion. While this inevitably introduces errors, a previous study by Streif et al (26) showed little influence of respiratory motion on T₁ mapping when the ratio of the breathing rate and the heart rate was low (about 1:8 in their study). This ratio was about 1:10 in our current study. Hence, the relatively low respiration rate (~50 cycles/min) in our study likely limited respiratory artifact beyond that which would be found when respiration occurs at a more physiological rate of 100–120 cycles/min. The extent to which the Mn²⁺ uptake is affected by this low respiratory rate needs further investigation.

The current method provides fast T₁ mapping via the use of saturation pulses, which eliminates the requirement of long waiting time to reestablish equilibrium magnetization. The time interval between the saturation modules (TR) was 20 R-R intervals, which covered the initial ascending portion of the recovery curve to up to ~2.4 s. In a previous study, we have evaluated the impact of the incomplete coverage of the saturation recovery curve on fitting accuracy by computer simulation (18). Our results suggest that a TR of 2.4 s is sufficient to yield accurate estimations for a wide range of T₁ values (0.2 to 1.7 s) that encompass possible myocardium T₁ changes from baseline to the end of contrast infusion in MEMRI experiments. While using longer TR can further improve the accuracy of T₁ estimation, the temporal resolution will be decreased.

To further improve the temporal resolution, only 64 lines at the center of k-space were acquired with one data average, leading to low spatial resolution and partial volume effects that will limit its ability to detect small areas with abnormal Ca²⁺ uptake. On the other hand, improving the spatial resolution to 128 phase-encoding lines will quadruple the acquisition time to achieve comparable SNR, leading to decreased temporal resolution that will not be sufficient to capture the dynamics of Mn²⁺-induced R₁ increase. Alternatively, the combination of the current method with other fast imaging methods, such as compressed sensing, can be used to provide both high temporal and spatial resolution.

Both dose groups showed progressive increase in R₁ during Mn²⁺ infusion. The increase in R₁ was relatively slow at the early stage of Mn²⁺ infusion, potentially because of the binding of Mn²⁺ to plasma proteins (2,19,27–29). With the increase of free Mn²⁺ concentration in plasma, a fast increase in R₁ occurred at the later stage of Mn²⁺ infusion. During Mn²⁺ washout, only a slight decrease in R₁ was observed, suggesting prolonged Mn²⁺ retention in cardiac myocytes (10,11). The two dose groups showed no difference in R₁ during the washout period. However, myocardial Mn²⁺ content was significantly higher in the high dose group (Fig. 7a). Previous studies have also reported an increase in Mn²⁺ accumulation in myocardium but similar R₁ increase at high Mn²⁺ infusion dose (13,19). These results suggest that T₁ shortening may saturate at high Mn²⁺ concentrations in myocardium.

The saturation of the T₁ shortening effect led to a plateau in R₁ increase at high Mn²⁺ concentration (Fig. 7b). Previously, Waghorn et al. reported the same saturation effect in mouse myocardium with a Mn²⁺ concentration above ~100 μg/g dry weight, or 0.47 mM using a wet-to-dry ratio of 4.05 (13,19), which is consistent with our current data. As a result, regression analysis using data from both dose groups resulted in an underestimation of Mn²⁺ relaxivity in myocardium by more than 50%. Using data only from the low dose group, the estimated Mn²⁺ relaxivity in myocardium is 6.02 mM⁻¹s⁻¹, which is in agreement with that reported in literature (4,13).

The current study also demonstrated the sensitivity of MEMRI to alterations in Ca²⁺ channel activity in mice deficient in α-dystrobrevin, i.e., the abdn^−/− mouse. The gene product, α-dystrobrevin, is a cytoplasmic protein of the dystrophin-glycoprotein complex. Grady et al. previously demonstrated the absence of sarcolemmal neuronal nitric oxide synthase (nNOS) associated with α-dystrobrevin disruption in adbn^−/− mice (22). Hence, the observed changes in the current study may be attributed to alterations in nitric oxide signaling on Ca²⁺ cycling. The effects of nNOS on calcium cycling have been investigated in nNOS-deficient (nNOS^−/−) mice previously(30–33). These in vitro studies demonstrated either elevated (31–33) or normal (30) basal Ca²⁺ current in nNOS^−/− myocytes. More recently, Vandsburger et al. also observed increased Mn²⁺ uptake in nNOS^−/− mice in vivo (16). Similar to the findings by Vandsburger et al., the adbn^−/− mice also showed a greater and faster R₁ increase compared to the wildtype mice (Fig. 8a), suggesting an increase in Ca²⁺ influx via the Ca²⁺ channels in myocardium, which was confirmed by the patch-clamp study in vitro (Fig. 8d&e). These data support an inhibitory role of sarcolemmal nNOS on Ca²⁺ channels.

The study on the adbn^−/− mice also suggests that the current method is sensitive to 16% increase in Ca²⁺ channel activity observed by the patch-clamp method. Numerous studies have observed a wide range of variations in Ca²⁺ channel activity in diseased hearts. Taking hypertensive hearts as an example, existing literature on Ca²⁺ channel activity has reported no change (34–36), up to more than 2-fold increase (37), or ~46% decrease (38). Whether the current method has the sensitivity to detect altered Ca²⁺ channel activity at different stages of the disease requires careful evaluation.

Using the current method, we did not observe a difference in Mn²⁺ uptake at apex, mid-ventricle and base in adbn^−/− mice, suggesting that changes in Ca²⁺ current are similar at different short-axis levels. The utility of the multi-slice T₁ mapping method would be better demonstrated in a mouse model with differences in Ca²⁺ channel activity at basal and apical levels. This pattern of Ca²⁺ channel alteration has been observed in a few disease models. In a rabbit model of drug-induced long QT type 2, the males showed a 22% increase in basal peak Ca²⁺ channel current as compared to the apex (39). In Takotsubo cardiomyopathy in humans, a transient wall motion abnormality of left ventricular apex that may lead to heterogeneous changes in Ca²⁺ channel activity (40). However, mouse models for these diseases have not been established.

Decreased heart rate was observed during Mn²⁺ infusion and subsequent washout (Fig. 6a). In contrast, a slight increase in ejection ratio was observed at the end of the imaging protocol (Fig. 6b). Previous studies on isolated perfused hearts (41) and papillary muscles (42) reported a negative inotropic effect of Mn²⁺ during the wash-in phase, followed by a positive inotropic effect during washout. While the decrease in heart rate during Mn²⁺ infusion and the slight recovery during the washout phase, as well as the increased ejection ratio at the end of Mn²⁺ washout observed in the current study, are consistent with these previous findings, one cannot rule out the possibility that prolonged anesthesia and increased blood volume due to Mn²⁺ infusion may also impact the hemodynamics of the heart. As such, further investigation is necessary to elucidate the impact of Mn²⁺ on cardiac inotropy and chronotropy.

In summary, an ECG-triggered, multi-slice saturation-recovery Look-Locker method was developed for fast cardiac T₁ mapping in mice. Validation studies showed strong agreement between the current method and the previously validated single-slice saturation-recovery Look-Locker method. The utility of MEMRI by MSRLL in detecting subtle changes in cellular Ca²⁺ current was evaluated in a study involving adbn^−/− mice. Our results suggest that fast T₁ mapping by MSRLL enabled the delineation of the kinetics of Mn²⁺ uptake in mouse myocardium, which allowed sensitive detection of a small increase in Ca²⁺ channel activity in adbn^−/− mice.