Abstract Body: Introduction: Transient Receptor Potential Melastatin 7 (TRPM7) is a cation channel bound to a kinase domain important for cellular Mg²⁺ homeostasis. We previously demonstrated that TRPM7 is cardiovascular protective and that TRPM7 downregulation and associated Mg²⁺ deficiency are associated with cardiovascular inflammation and fibrosis. Here, we investigated underlying molecular mechanisms by studying hearts, cardiac fibroblasts (CF) and small arteries from wild-type (WT) and TRPM7-deficient mice (M7+/Δ) focusing on calpain, a downstream target of TRPM7 kinase.
Methods: Hearts and primary culture CF from WT and M7+/Δ mice were used to probe intracellular signaling pathways and pro-fibrotic proteins. Mg²⁺ influx was assessed by fluorescence. Protein expression was assessed by immunoblotting. To evaluate direct vascular effects of TRPM7 activation, we studied effects of naltriben, a TRPM7 activator in vessels exposed to low Mg²⁺ conditions (0.2mM), to mimic M7+/Δ. Vascular function was assessed by wire myography.
Results: Hearts from M7+/Δ had increased collagen deposition (3.3% vs WT: 1.6%). CF M7+/Δ showed reduced influx of Mg²⁺ (106% vs WT 115%) and increased expression of TGFβ1 (2.14±0.38 vs WT 0.25±0.19) and phospho-SMAD3 (1.21±0.06 vs WT 0.72±0.05). This was associated with increased expression of fibronectin in CF M7+/Δ vs WT (0.99±0.18 vs 0.42±0.12), α-SMA (1.51±0.13 vs 0.55±0.99) and periostin (1.33±0.24 vs 0.42±0.09). Expression of TGFβ1 and phospho-SMAD3 was normalized by Mg²⁺ treatment. CF M7+/Δ exhibited increased calpain-2 (1.55±0.22 vs WT 0.55±0.07) and reduced annexin-A1 (0.56±0.04 vs WT 1.40±0.08), known target proteins of TRPM7-kinase. Treatment with calpastatin (10^-5M), a calpain inhibitor, normalized expression of TGFβ1, fibronectin and α-SMA. Low Mg²⁺ treated vessels exhibited increased contraction induced by U46619 (Emax% 348 vs veh 203) and reduced sensitivity to relaxation by Ach (LogEC50 6.1 vs veh 6.8) and SNP (LogEC50 5.9 vs veh 6.5). Naltriben ameliorated contraction and SNP-relaxation (p<0.05).
Summary and Conclusion: Upregulation of cardiac pro-fibrotic and pro-inflammatory processes in M7+/Δ was ameliorated by Mg²⁺ and calpastatin. Vascular dysfunction in Mg²⁺-deprived vessels was normalized by TRPM7 activation. Together our findings indicate that cardiovascular injury in TRPM7-deficient states involves calpain and that Mg²⁺/TRPM7 activation is cardiovascular protective.