Background Translocation of miR-181c into cardiac mitochondria downregulates the mitochondrial gene, mt-COX1. miR-181c/d^-/- hearts experience less oxidative stress during ischemia/reperfusion (I/R) and are protected against I/R injury. Additionally, miR-181c overexpression can increase mitochondrial matrix Ca²⁺ ([Ca²⁺]_m), but the mechanism by which miR-181c regulates [Ca²⁺]_m is unknown. Methods and Results By RNA sequencing and analysis, here we show that hearts from miR-181c/d^-/- mice overexpress nuclear-encoded Ca²⁺ regulatory and metabolic pathway genes, suggesting that alterations in miR-181c and mt-COX1 perturb mitochondria-to-nucleus retrograde signaling and [Ca²⁺]_m regulation. Quantitative polymerase chain reaction validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 (specificity protein) expression in the miR-181c/d^-/- hearts. Furthermore, an association of Sp1 with the promoter region of MICU1 was confirmed by chromatin immunoprecipitation-quantitative polymerase chain reaction and higher expression of MICU1 was found in the miR-181c/d^-/- hearts. Conversely, downregulation of Sp1 by small interfering RNA decreased MICU1 expression in neonatal mouse ventricular myocytes. Changes in PDH activity provided evidence for a change in [Ca²⁺]_m via the miR-181c/MICU1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU1 was knocked down in the miR-181c/d^-/- heart by lentiviral expression of a short-hairpin RNA against MICU1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an in vitro I/R model in miR-181c/d^-/- neonatal mouse ventricular myocytes, we confirmed the contribution of both Sp1 and MICU1 in ischemic injury. Conclusions miR-181c regulates mt-COX1, which in turn regulates MICU1 expression through the Sp1-mediated mitochondria-to-nucleus retrograde pathway. Loss of miR-181c can protect the heart from I/R injury by modulating [Ca²⁺]_m through the upregulation of MICU1.

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