The role of transient receptor potential melastatin channels in compressive force‐induced contraction of primary cardiac pericytes

Figures

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  FIGURE 1. Confocal immunocytochemistry of primary cardiac pericytes. (a) Primary cardiac pericytes exhibit a single, uniform morphology in culture as demonstrated by confocal differential interference contrast imaging (gray). Primary cardiac pericytes also maintain expression of pericyte markers CD‐146 (b), PDGFRβ (c), NG2 (d), α‐SMA (e), and Vimentin (f) shown by immunocytochemistry overlaid with F‐actin marker Phalloidin (red). Images in the bottom panel show Hoechst 33342 staining for nuclei. Scale bar = 20 μm.

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  FIGURE 2. TRPM4 and TRPM7 are expressed in primary cardiac pericytes. (a) Global RNAseq from 3 separate cultures identified four TRP channels present in primary cardiac pericytes: TRPM7, TRPM4, TRPC2, and TRPC1. Expression is plotted as a function of detected Log2CPM relative to β‐actin. (b) Relative mRNA abundance was validated by quantitative PCR, with TRPM7 and TRPM4 again detected at highest abundance. Expression is plotted as a function of 1/cycle threshold (CT) relative to β‐actin. (c) Immunoblotting cellular lysates of primary cardiac pericytes demonstrates protein expression of TRPM7 and TRPM4. (d) Immunocytochemistry demonstrates TRPM4 and TRPM7 expression. TRPM4 appears to strongly localize in perinuclear networks, while TRPM7 appears diffusely expressed throughout the membrane. Scale bar = 20 μm. (e) IHC of heart slices from transgenic mice expressing DsRed under the pericyte‐specific NG2 promoter demonstrates TRPM4 and TRPM7 expression in the coronary vasculature of mice. Both TRPM4 and TRPM7 are colocalized in DsRed positive cells with the size and morphological characteristics consistent with pericytes (arrows). Some expression is also observed in non‐vascular cells…

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  FIGURE 3. Pharmacological activation of TRPM4 or TRPM7 mobilizes Ca2+ in primary coronary pericytes. (a) Workflow for agonist induced Ca2+ mobilization in Fluo‐4 AM loaded cultures. AUC for a 1 min increment immediately following bath injection of TRPM4 agonist BTP2 or TRPM7 agonist Naltiben is compared to the AUC prior to any activity. (b) Representative readout of calcium fluorescence before, during, and after an agonist stimulation demonstrating a prominent increase in internal calcium fluorescence within seconds of agonist injections. (c, left panel) The TRPM4 agonist BTP2 (100 nM) induced strong Ca2+ mobilization when pre‐treated with vehicle alone (87.8 ± 19.9%, n = 11). Pretreatment with the TRP channel modulator Ruthenium Red had no significant impact on this effect (10 μM, 85.4 ± 33.1%, p = 0.99, n = 7), while pre‐incubation with TRPM4 antagonists Flufenamic acid (50 μM) and 9‐Phenanthrol (1 μM) significantly decreased this effect (29.8 ± 12.7%, n = 12; and 24.9 ± 8.5%, n = 11, respectively). (c, right panel) The TRMP7 agonist Naltriben (100 nM) also caused an increase in Ca2+ when pre‐treated with vehicle alone (122 ± 19.2%, n = 10). Pretreatment with Ruthenium Red caused no…

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  FIGURE 4. TRPM4 and TRPM7 mediate Ca2+ mobilization in primary cardiac pericytes induced by compressive force. (a) Depiction of compressive force application. Cultured primary glass pericytes in glass bottom dishes are loaded with Fluo‐4 AM. Then a thin glass coverslip is slid overtop to allow calibration weights to gently apply a compressive force without visible damage to cellular integrity. (b) Workflow for experimental series. Area under the curve (AUC) for 1 min immediately following compressive force is compared to a 1 min AUC of no activity. (c) Representative readout of Ca2+ fluorescence before, during, and after a compressive force experiment. Compressive force induces a clear Ca2+ mobilization without tearing or lifting cells. (d) Compressive force induces an immediate increase in Ca2+ mobilization in vehicle treated plates (ΔF/F = 100 ± 11%, n = 14) that is not significantly altered by pre‐incubation with Ruthenium Red (10 μM, 120 ± 15.9%, n = 9) or TRPC antagonist SKF96365 (10 μM, 73.5 ± 10.3%, n = 7). Ca2+ mobilization induced by compressive force was, however, significantly decreased by pre‐incubation with antagonists to TRPM4 (Flufenamic Acid: 50 μM, 53.3 ± 12.4%, n = 11;…

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  FIGURE 5. TRPM4 Blocker 9‐Phenanthrol markedly reduces infarct size in mouse model. Infarct size data from animals treated with vehicle (n = 8) and 9‐phenanthrol (n = 7). A large infarct (pale tissue unstained with TTC and marked by white arrows) is noted in the vehicle treated animal (a) while the infarct size in a 9‐phenanthrol treated animal is much smaller. The risk area is represented by tissue not stained with Evan's Blue. Aggregate data (b) shows a significant difference in infarct size/risk area ratio between the two groups of animals. There is a 3.5‐fold reduction in the infarct size/risk area ratio in 9‐phenanthrol treated compared to vehicle treated animals (9.4 ± 9.7% vs. 32.9 ± 28.9%). Red denotes females and blue denotes males.