Loss of MD1 exacerbates pressure overload-induced left ventricular structural and electrical remodelling

Myeloid differentiation protein 1 (MD1) has been implicated in numerous pathophysiological processes, including immune regulation, obesity, insulin resistance, and inflammation. However, the role of MD1 in cardiac remodelling remains incompletely understood. We used MD1-knockout (KO) mice and their wild-type littermates to determine the functional significance of MD1 in the regulation of aortic banding (AB)-induced left ventricular (LV) structural and electrical remodelling and its underlying mechanisms. After 4 weeks of AB, MD1-KO hearts showed substantial aggravation of LV hypertrophy, fibrosis, LV dilation and dysfunction, and electrical remodelling, which resulted in overt heart failure and increased electrophysiological instability. Moreover, MD1-KO-AB cardiomyocytes showed increased diastolic sarcoplasmic reticulum (SR) Ca2+ leak, reduced Ca2+ transient amplitude and SR Ca2+ content, decreased SR Ca2+-ATPase2 expression, and increased phospholamban and Na+/Ca2+-exchanger 1 protein expression. Mechanistically, the adverse effects of MD1 deletion on LV remodelling were related to hyperactivated CaMKII signalling and increased impairment of intracellular Ca2+ homeostasis, whereas the increased electrophysiological instability was partly attributed to exaggerated prolongation of cardiac repolarisation, decreased action potential duration alternans threshold, and increased diastolic SR Ca2+ leak. Therefore, our study on MD1 could provide new therapeutic strategies for preventing/treating heart failure.


Surface Electrocardiogram (ECG) recording and analysis
Surface-lead ECG (lead II) recording was performed on mice under light anesthesia. Mice were lightly anesthetized by inhaled isoflurane (1.5% isoflurane in 98% O2) and were positioned on a custom-made ECG recording platform, body temperature was maintained at 37°C by use of a heating pad controlled by a temperature controller (World Precision Instruments). The Ag/AgCl gel-coated ECG electrodes were placed subcutaneous and connected to a standard 6-lead ECG amplifier module (AD Instruments, Australia), which included high and low pass filters (set to 0.05 Hz and 1kHz, respectively) and a gain selection device (set to 1000-fold). ECG Signals were continuously recorded at 1 kHz sampling rate using the data acquisition system (AD Instruments) for 30 min. Data were analyzed off-line using LabChart 7 Pro (AD Instruments). After scanning the ECG signals for rhythm disorders and noise, a stable periods of 5 min were analyzed to determine RR, PR, QRS and corrected QT (QTc) intervals. The RR interval was determined automatically by averaging the time between two consecutive RR waves. The PR interval was measured from the beginning of the P wave to the beginning of the QRS complex. QRS duration was measured from the first deflection of the Q wave (or the R wave when the Q wave was absent) to the point where the negative part of the S wave returned to the isoelectric line. The QT interval was measured from the beginning of the QRS complex to the end of the T wave. To correct for heart rate, QTc interval was calculated with Bazett's formula 2 : QTc = QT/(RR/100) 1/2 .

Preparation of Langendorff-perfused hearts
The isolated Langendorff-perfused hearts were prepared according to published methods 3 . Mice were heparinized by heparin sodium (100U, intraperitoneal injection) for 10 min, then anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) and adequacy of anesthesia was monitored by testing the pedal reflex. Hearts were quickly excised and transferred to oxygenated and ice-cold Tyrode's solution (mmol/L: NaCl 135; KCl 5.4; CaCl2 1.8; MgCl2 1; NaH2PO4 0.33; HEPES 10; glucose 10; pH adjusted to 7.35 with NaOH). The ascending aorta was identified and cannulated with a tailor-made 21-gauge cannula that had been prefilled with ice-cold buffer, make sure the terminal of the gauge cannula was located above of the aortic root. The aorta was secured onto the cannula with a microaneurysm clip. The heart was then rapidly transferred and fixed to the langendorff-perfusion system (AD Instruments). The oxygenated perfusate was passed through the pipeline and warmed to 37°C by a water jacket and circulator, then through the aorta at 2-3 ml/min by a peristaltic pump (AD Instruments, Australia).The perfusion pressure was maintained at 80-100mmHg. By this way, the coronary arteries were perfused with oxygenated Tyrode's solution passing through the aorta. After the initiation of perfusion, hearts regained a pink color and spontaneous rhythmic contractions. All the isolated hearts were perfused for more than 10 min before further experiments. The hearts that did not recover to regular spontaneous rhythm or had irreversible myocardial ischemia were discarded.

Monophasic action potential (MAP) recording
MAP was recorded from the epicardium of the LV anterior free wall using a custom-made MAP electrode, constructed from two 0.25 mm Teflon-coated silver wire (99.99% purity) which were twist together and galvanically chlorided to eliminate DC offset. The paired platinum stimulating electrode was positioned on the basal surface of right ventricle and delivered regular pacing. MAPs were amplified with an amplifier and band pass filtered between 0.3 Hz and 1 kHz. MAP waveforms were analyzed using LabChart 7 Pro software.

Preparation of mouse LV myocytes
Mice were heparinized and anesthetized as described above, the hearts were quickly removed and placed into a cold and oxygenated Ca 2+ -free Tyrode's solution containing (in mmol/L) NaCl 135, KCl 5.4, MgCl2 1, NaH2PO4 0.33, HEPES 10, glucose 10, adjusted to pH 7.35. The ascending aorta was cannulated onto the langendorff-perfusion system at a 2-3 ml/min flow rate of perfusion. The heart was perfused with oxygenated Ca 2+ -free Tyrode's solution at 37°C for 5-10 min to flush away any blood inside the heart. For enzymatic dissociation, the heart was perfused with oxygenated Ca 2+ -free Tyrode's solution containing collagenase type II (0.3-0.5 mg/ml; Sigma Aldrich, C6885) and BSA (2 mg/ml; Roche Diagnostics, 10735086001) for 10-15 min at 37°C. Then the heart was removed and placed into a dish containing Tyrode's solution supplemented with 0.1 mmol/L CaCl2 and 2 mg/ml BSA. The left ventricle was separated from the heart, cut into small pieces, and triturated with a pipette to disperse the myocytes. Ventricular myocytes were filtered on gauze and sedimented by gravity for 10 min. The supernatant was removed, and cells were suspended in Tyrode's solution containing 0.3 mmol/L CaCl2 and 2 mg/ml BSA. The procedure was repeated once, and cells were suspended in Tyrode's solution containing 0.5 mmol/L CaCl2 and 2 mg/ml BSA. Freshly isolated LV myocytes were stored at room temperature until use. Only rod-shaped myocytes showing clear striations were studied, and experiments were performed at room temperature (20-25°C) within 6 h after cell isolation.

Patch-clamp recording
The whole-cell patch-clamp technique was used to record L-type Ca 2+ channel current (ICa, L). Patch electrodes were pulled from borosilicate glass (Sutter Instrument, BF150-86-10) using six-stage pulling on a Flaming/Brown Micropipette puller (Sutter Instrument, Model P-97). The fire-polished electrodes had a resistance of 4 to 8 MΩ when filled with internal solution contained (in mmol/L): CsCl 120, EGTA 11, CaCl2 1 , MgCl2 5, Na2-ATP 5, HEPES 10, glucose 11, titrated to pH 7.35 with CsOH. The cells were continuously perfused with extracellular solution contained (in mmol/L): NaCl 35, Choline chloride 100, glucose 10, CaCl2 1.8, HEPES 10, MgCl2 1, KCl 5.4, NaH2PO4 0.33, BaCl2 0.1, 4-aminopyridine 5, titrated to pH 7.35 with NaOH. ICa, L activation was measured by applying a 300-ms pulses of voltages between -50 mV and +60 mV in 10 mV steps preceded by a 100-ms prepulse of -50 mV. The steady state inactivation of ICa, L was measured by applying a 300-ms prepulse of potentials from -50 to +60 mV in 10 mV steps, followed by a fixed 300-ms test pulse of +20 mV. Whole cell membrane currents were obtained and assessed with an EPC-9 patch-clamp amplifier (HEKA Electronik, Lambrecht, Germany) in the whole-cell mode by the Pulse/Pulsefit software program. The nonlinear curve fitting of inactivation of ICa,L was performed with Origin 9.0 (OriginLab Co. USA) using the Boltzmann equation.    (C) peak current density for ICa, L of the indicated groups (cells/mice, n = 12-13/5-6). (D) Mean values for steady-state inactivation of I Ca, L and (E) bar diagram of half-maximal potential (V 1/2) for I Ca, L inactivation of the four groups (cells/mice, n = 12-13/5-6). The pulse protocol is inset in Fig. 5D. Values were fitted t o the Boltzmann equation and were analysed with PEMS software. *P < 0.05 vs. WT-Sham, § P < 0.005 vs. MD1-KO-Sham.