Wt (C57BL/10SnJ) and mdx (CS7BL/10ScSn-mdx) male (6 months old) mice were obtained from breeding colonies at the Mount Sinai Medical Center, from founders initially obtained from the Jackson Laboratory (Bar Harbor, ME). All protocols used in the study were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the institution (IACUC Protocol #19090).

Mdx and their Wt non-dystrophic littermates were euthanized using CO₂ or cervical dislocation. VSMCs were isolated using a modification of a previously described method (Ray et al., 2001). In brief, the aorta was dissected from its origin at the left ventricle to the iliac bifurcation, and the vessel was cut and placed in Hank’s Balanced Salt Solution (HBSS) (Thermo Fisher Scientific, FL, United States). Using a dissecting microscope, the fat tissue and the adventitia were removed; then, the aorta was irrigated with HBSS plus 2.5 μg/mL Fungizone (Thermo Fisher Scientific, FL, United States). The vessel was open longitudinally, and with sterile cotton swabbed, the endothelial layer of cells was gently removed and then cut into small segments (around 4 mm² each). The aorta segments were incubated in HBSS containing 1 mg/ml collagenase type 2 (Worthington Biochemical Corporation, NJ, United States) for 30 min. Then, the solution containing collagenase type 2 was replaced with a solution containing 1 mg/ml collagenase type 2 and 0.5 mg/ml elastase (Worthington Biochemical Corporation, NJ, United States). After the second digestion step, the remaining tissue was mechanically dissociated in the dish by flushing through a series of decreasing size fire-polished pipettes. Fresh HBSS was then added to stop the enzymatic digestion, and the cell suspension was centrifugated at 600 × g. The cell pellet was resuspended and centrifugated again at 600 × g and then transferred to a Matrigel-coated 24-well cell culture plate containing smooth muscle cell growth medium (SGM-2, Lonza, GA, United States). Isolated VSMC were cultured in a humidified atmosphere (37°C) and for 7–10 days after platting before experimentation.

The following criteria were used to judge the functionality of VSMCs: (i) no cell shortening was observed when they perfused with the Ca²⁺ containing Ringer solution (1.8 mM Ca²⁺) and (ii) they contracted in response to electrical stimuli (1 ms square pulse duration, ∼1.5 × threshold voltage).

Double-barreled Ca²⁺ and Na⁺ selective microelectrodes were prepared as described previously (Eltit et al., 2013). Single smooth muscle cells were impaled with either a Ca²⁺ – or Na⁺-selective double-barreled microelectrode, and their potentials were recorded via a high-impedance amplifier (WPI Duo 773 electrometer; World Precision Instruments, FL, United States). Criteria for successful impalement of single muscle cells included an (i) abrupt drop to a steady level of Vm more negative than −55 mV, (ii) a recording stable for both potentials (Vm and Ca potential) for at least 60 s and (iii) an quick return to baseline on the exit of the microelectrodes from the cell. The specific Ca^{2 +} potential (V_Cae) or Na⁺ potential (V_Nae) was obtained by subtracting the V_Ca potential or V_Na from the 3 M KCl microelectrode potential (Vm); Vm, and the specific Ca²⁺- Na⁺ potentials were stored in a computer for future analysis.

VSMCs were seeded on flexible-bottomed culture plates coated with poly-L-lysine (Flexcell International Corp., NC, United States). After 48 h to allow for cell attachment and spreading, Wt and mdx VSMCs were bathing with Ringer solution and then subjected to mechanical stretch elongation of 30 cycles/min (0.5 Hz), 20% elongation using a Flexcell FX 5000 tension system for 5 min. After the cyclic stretch, to estimate cell damage, the medium was collected for the determination of lactate dehydrogenase (LDH) activity (released by VSMCs) using the LDH kit from Sigma-Aldrich (St. Louis, MO, United States) according to the manufacturer’s instructions. Parallel series of Flex culture plates not subjected to stretching served as controls. At the time of collection of the medium for LDH determination, [Ca²⁺]_i or [Na⁺]_i was measured in the stretched VSMCs using ion-selective microelectrodes.

Mdx and Wt anesthetized mice were euthanized using CO₂. The aorta was dissected as described above (Primary culture of VSMC). Aortic total protein extraction was performed using a modified Millipore enzyme buffer with added 0.5% Triton-X-100 for 1 h digestion and lysis step. After proteins transfer from the gel, we spliced the membrane horizontally according to the molecular weight of proteins of interest. Then, individual membrane strips were incubated with the primary anti-TRPC1, TRPC3, TRPC6, and dystrophin antibodies (Uryash et al., 2015). The corresponding protein size was determined based on the protein standard marker. The levels of target protein(s) were normalized to loading control using the housekeeper protein β-actin.

The normal Ringer’s solution contained the following (in mM): 135 NaCl, 5 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 glucose, 3.6 NaHCO₃ (pH 7.4). In all experiments, Wt and mdx VSMCs were perfused and equilibrated with the Ringer’s solution aerated with a mixture of 95% O₂ and 5% CO₂ at 37°C. For the Ca²⁺ free solution CaCl₂ was omitted and 2 mM MgCl₂ and 1 mM EGTA were added in its place. 1-oleoyl-2-acetyl-sn-glycerol (OAG) (100 μM) a TRPC3, and TRPC6 channel activator, SAR7334 (0.1 and 1 μM) a TRPC3 and TRPC6 channel blocker, GsMTx4 (5 μM) a specific mechanosensitive channel inhibitor, nifedipine (10 μM) a selective voltage-gated Ca²⁺ channel blocker solutions were prepared from stock solutions.

All values are reported as mean ± SD. Student’s t-test or analysis of variance (1-way ANOVA and Tukey’s post hoc tests were used to determine significance. p < 0.05 was considered statistically significant. n_mice: number of mice used experimentally, n_cell: represents the number of successful measurements carried out.

Striated muscle cells from DMD patients and mdx mice (Lopez et al., 1987; Altamirano et al., 2013, 2014; Mijares et al., 2014) show elevated intracellular Ca²⁺ and Na⁺. Therefore, intracellular [Ca²⁺] and [Na⁺] were measured in VSMCs isolated from Wt and mdx mice using double-barreled ion-specific microelectrodes. [Ca²⁺]_i was elevated in mdx VSMCs (266 ± 27 nM, n = 45) compared to that observed in Wt cells (121 ± 3 nM, n = 41), (p < 0.001) (Figure 1). Similarly, [Na⁺]_i was higher in mdx VSMCs (14 ± 1.2 mM n = 25) than observed in Wt cells (8 ± 0.1 mM n = 25) (p < 0.001) (Figure 1).

To directly investigate the effect of diacylglycerol (DAG) on [Ca²⁺]_i and [Na⁺]_i in smooth muscle cells, VSMCs were exposed to the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) which activates TRPC3 and TRPC6 channels (Hofmann et al., 1999). Incubation in OAG (100 μM) for 10 min produces an elevation of [Ca²⁺]_i and [Na⁺]_i in both genotypes. Figures 2A–D show representative measurements of the resting membrane potential (Vm) and [Ca²⁺]_i from Wt and mdx VSMCs before and after exposure to OAG. In Wt VSMCs OAG provoked an increase of [Ca²⁺]_i by 46%, from 121 ± 3 nM, n = 22, to 177 ± 19 nM, n = 25 (p < 0.001), and in mdx VSMCs by 73%, from 271 ± 29 nM, n = 25, to 470 ± 55 nM, n = 27 (p < 0.001) (Figure 3A). [Na⁺]_i was also significantly elevated in Wt by 25% and in mdx VSMC by 46% upon incubation in OAG (Figure 3B). We examined the possible involvement of the voltage-gated Ca²⁺ channels in the OAG-induced elevation of [Ca²⁺]_i by pretreating cultured VSMCs with nifedipine 10 μM, a specific voltage-gated Ca²⁺ channel blocker. Nifedipine had no significant effects on OAG-induced elevation of [Ca²⁺]_i in either Wt or mdx VSMCs (Supplementary Figure S1).

To establish the impact of extracellular Ca²⁺ ([Ca²⁺]_e) on OAG-induced elevation of [Ca²⁺]_i, Wt and mdx VSMCs were incubated for 5 min in Ca²⁺-free solution before OAG (100 μM) treatment. Exposure to reduced [Ca²⁺]_e significantly lowered [Ca²⁺]_i in Wt and mdx VSMCs but had a greater effect in mdx (−49%, from 271 ± 24, n = 15 to 138 ± 11, n = 18, p < 0.001) than in Wt (−18%, from 122 ± 3 nM, n = 15 to 100 ± 5 nM, n = 17, p < 0.001) VSMCs (Figure 4). In the absence of [Ca²⁺]_e, the previously observed rise in [Ca²⁺]_i elicited by OAG in Ca²⁺ replete buffer was completely inhibited in both Wt and mdx VSMCs (Figure 3). In addition to the decrease in [Ca²⁺]_i, incubation in Ca²⁺-free solution leads to a reversible depolarization of cell membrane potential in both genotypes of about 4–6 mV despite the presence of 2 mM Mg²⁺ (Supplementary Figure S2).

To gain insight into molecular mechanisms resulting in [Ca²⁺]_i elevation upon exposure to OAG, we measured [Ca²⁺]_i in Wt and mdx VSMCs before and after incubation with SAR7334 which is a blocker of TRPC6 and TRPC3 channels (Maier et al., 2015), and then again after exposure to OAG (100 μM). Pretreatment with SAR7334 significantly lowered [Ca²⁺]_i in dose-dependent manner in both genotypes. Pretreatment with 0.1 μM SAR7334, a concentration that block mostly TRPC6 channels (Maier et al., 2015) reduced [Ca²⁺]_i in Wt by 6% and in mdx VSMCs by18% (Figure 5A). Preincubation with SAR7334 (1 μM) that blocks TRPC3 and 6 channels (Maier et al., 2015) provoked further reduction of [Ca²⁺]_i in Wt (15%) and mdx VSMCs (50%) (Figure 5B). The Figure 6 shows representative records of the effects of SAR7334 on [Ca²⁺]_i in Wt VSMCs (Figure 6B), and mdx VSMCs (Figure 6D). In addition, SAR7334 1 μM also reduced [Na⁺]_i in Wt (13%) and in mdx VSMCs (35%) (Figure 5C) and prevented any significant increase in [Ca²⁺]_i and [Na⁺]_i upon exposure to OAG in both genotypes (Figures 5B,C).

Stretching smooth muscle cells has previously been shown to increase [Ca²⁺]_i (Zou et al., 2002; Ducret et al., 2010). Numerous members of the TRPC channel family, especially TRPC1, TRPC3, and TRPC6 are considered to be mechanosensitive channels (Friedrich et al., 2012; Takahashi et al., 2013) and are therefore possible candidates for this increase. In our VSMC stretch experiments [Ca²⁺]_i and [Na⁺]_i increased in both genotypes; however, the magnitude of the increases in Ca²⁺ and Na⁺ were greater in mdx than Wt. In Wt VSMCs [Ca²⁺]_i was elevated by 39% from 121 ± 3 nM, n = 25 to 169 ± 18 nM, n = 23 (p < 0.001) (Figure 7A) and [Na⁺]_i by 31% from to 8 ± 0.1 mM, n = 10 to 11 ± 1 mM, n = 10 (p < 0.001) (Figures 7B). In mdx VSMCs [Ca²⁺]_i was elevated by 69% from 285 ± 25 nM, n = 25 to 482 ± 37 nM, n = 20 (p < 0.001) (Figure 7A) and [Na⁺]_i by 43% from to 14 ± 1.2 mM, n = 19 to 20 ± 1.8 mM, n = 12 (p < 0.001) (Figure 7B). To test whether the elevation of [Ca²⁺]_i associated with stretch was mediated by Ca²⁺ influx through sarcolemma Ca²⁺ channels, extracellular Ca²⁺ was removed and 2 mM MgCl₂ and 1 mM EGTA were added to the bathing supernatant (see section “Materials and Methods”). Under these conditions the increase in [Ca²⁺]_i in response to stretch was abolished in both Wt and mdx VSMCs (Figure 8A). Re-addition of extracellular Ca²⁺ before repeating the stretch protocol allowed recovery of the increase in both genotypes. These results suggest that a Ca²⁺ influx was involved in the elevation of [Ca²⁺]_i upon the mechanical stretch. To further dissect the mechanism involved in the stretch-induced elevation of [Ca²⁺]_i in VSMCs we tested the effect of GsMTx-4 (5 μM), which is known to inhibit mechanosensitive channels (Spassova et al., 2006; Bowman et al., 2007). GsMTx-4 completely inhibited the stretch-induced increases of [Ca²⁺]_i in both genotypes (Figure 8B). Additionally, we examined whether the stretch-induced elevation of [Ca²⁺]_i was mediated via L-type voltage-gated Ca²⁺ channels by incubating the VSMCs with the Ca²⁺ channel blocker nifedipine (10 μM). The stretch-induced increase in [Ca²⁺] was not modified by nifedipine in either genotype (Supplementary Figure S3).

Resting LDH activity in the supernatant from non-stretched mdx VSMCs (a marker of cell damage) was 35% greater than in the supernatant from Wt VSMCs (Figure 9). Muscle stretching increased LDH activity in both genotypes; however, the increase was more significant in mdx than Wt VSMCs (41% in Wt vs. 90% in mdx VSMCs) (Figure 9).

To determine whether the elevation of [Ca²⁺]_i and [Na⁺]_i and enhanced response to OAG observed in dystrophic VSMs was associated with changes in TRPC protein in the membrane, the expression of TRPC1, -3 and -6 were measured using Western blot analysis. Analysis of these blots demonstrated that TRPC1, TRPC3, and TRPC6 were significantly upregulated in VSMCs from mdx compared to Wt mice (Figures 10A,B and Supplementary Figure S4).

Despite the novelty of our study, some limitations should be acknowledged. First, we used a pharmacological approach to characterize the involvement of TRPC channels in the dysregulation of intracellular Ca²⁺ observed in VSMCs from mdx mice, and we did not study the functional aspects of TRPC channels. Secondly, we did not carry out experiments in which TRPC1, -3, and -6 channels were individually or collectively downregulated using siRNA. Therefore, we were unable to assess whether decreasing TRPC channel expression rescues or improves intracellular Ca²⁺ regulation in mdx VSMCs.