Discussion

The resolution of inflammation and subsequent tissue repair after MI hinge on the pivotal transition of cardiac macrophages toward a reparative phenotype, a cell population indispensable for modulating local immune responses in the heart [35]. The phenotype of macrophages is closely related to the metabolic pathway [36]. MTT is an emerging approach for the treatment of diseases with mitochondrial dysfunction such as MI [31]. However, the clinical translation of MTT is severely constrained by three interrelated bottlenecks: the fragility of mitochondria, the hostile post-MI microenvironment, and the low efficiency of mitochondrial internalization by recipient cells [12,15,16,37]. Here, inspired by the intrinsic cellular defense mechanism against mitochondrial dysfunction, we reported MQgel@Mito, a mitochondria-derived peptide hydrogel system encapsulating functional mitochondria. This system substantially extended organelle viability, enhanced internalization by macrophages, and promoted cardiac repair through macrophage metabolic reprogramming.

Different from conventional mitochondrial delivery systems that rely merely on passive physical protection, the superior protective capacity of MQgel for mitochondria stems from the combination of physical shielding and intrinsic bioactive effects. Mechanistically, the hydrogel matrix serves as a protective barrier that insulates encapsulated mitochondria from detrimental extracellular microenvironments [17,38]. Additionally, the hydrogel matrix, with its porous three-dimensional architecture and highly hydrated environment, prevents mitochondrial aggregation and maintains mitochondrial dispersion, ensuring efficient exchange of metabolic substrates and byproducts to sustain mitochondrial metabolic activity [18,39]. Beyond passive physical protection, MQgel integrates the mitochondrial-derived peptide MOTS-c and exhibits inherent bioactivity capable of stabilizing mitochondrial energy metabolism and eliminating excessive ROS [26]. Our data revealed that 30 mg mL−1 MQgel extended this window to 8 h, a remarkable improvement that transformed MTT from a logistically constrained procedure into a clinically tractable intervention. Besides, the donor mitochondria are transplanted to a harsh transplantation environment with excess oxidative stress and calcium overload following MI [17,40]. Our findings demonstrated that MQgel-encapsulated mitochondria exhibited markedly preserved ATP synthesis ability, respiratory chain function, and mitochondrial membrane potential following exposure to these adverse factors. These protective effects are particularly consequential during the critical period between intramyocardial delivery and cellular internalization, when naked mitochondria would otherwise be vulnerable to environmental insult.

Donor mitochondria should be internalized by recipient cells in order to exert their functions [9,34,41]. A central finding of this work is that the MQ enhanced mitochondrial internalization through an AMPK-dependent mechanism. As reported, mitochondria can be internalized via macropinocytosis [16]. As a cellular energy sensor, AMPK pathway can drive cytoskeletal remodeling by activating the actin-severing protein cofilin, thereby enhancing the formation of macropinosome [[27], [28], [29]]. Therefore, our results suggested that MQ facilitated the internalization of isolated mitochondria by macrophages through AMPK-driven macropinocytosis. The observed colocalization of donor mitochondria with the recipient mitochondrial network suggested functional integration rather than lysosomal degradation, which was a critical consideration for sustained therapeutic benefit.

Unlike previous studies focusing on direct cardiomyocyte protection, our work focuses on the immunometabolic effects of MQ@Mito and its contribution to cardiac tissue regeneration post MI. Macrophage polarization is intimately coupled to cellular metabolism: pro-inflammatory macrophages rely upon glycolysis with impaired OXPHOS, whereas pro-reparative macrophages depend upon mitochondrial function [36]. Glycolysis provides a rapid but less efficient ATP yield (∼2 per glucose), whereas an intact TCA cycle maintains a sustained supply of ATP (∼30 per glucose) through OXPHOS and fatty acid oxidation [42]. LPS disrupts the TCA cycle by inhibiting the expression and activity of isocitrate dehydrogenase, thereby causing the accumulation of isocitrate and its upstream metabolite, cis-aconitate [43]. Our metabolomic and functional analyses revealed that by providing functionally intact mitochondria, MQ@Mito treatment suppressed LPS-induced glycolysis while maintaining TCA cycle integrity and enhancing OXPHOS capacity. This metabolic reprogramming was accompanied by phenotypic changes consistent with attenuated pro-inflammatory M1 polarization and enhanced M2 polarization. The mechanistic link between bioenergetic state and inflammatory phenotype underscored the potential of metabolism-targeted interventions for modulating macrophage function (Fig. 5P).

In vivo, MQgel@Mito prevented the rapid decline in myocardial contractility following MI, resulting in a marked improvement in cardiac function, inhibited adverse remodeling, and alleviated thinning of LV wall. The efficacy of MQgel@Mito for MI treatment substantially exceeded that of either MQgel or mitochondrial transplantation alone. MQgel protected donor mitochondria against the hostile transplantation environment with oxidative stress and calcium overload in the infarct border zone and thereby enhanced the function of donor mitochondria. The controlled degradation profile of MQgel enabled sustained mitochondrial release rather than burst delivery, optimizing the absorption and utilization of organelles during the critical early post-MI period. Macrophages are one of the most active cell types across all post-MI stages, including the inflammatory, cardioprotective, and cardiac repair phases [4]. Macrophage-targeted therapy plays a pivotal role in post-MI recovery. We found that MQgel@Mito inhibited the infiltration of M1-like macrophages, decreased secretion of pro-inflammatory cytokines, and increased secretion of reparative cytokines, thereby significantly mitigating excessive inflammatory response after MI. The mechanism of anti-inflammation was closely associated with the metabolic reprogramming of macrophages induced by MQgel@Mito. Our transcriptomic analysis provided molecular confirmation of the observed functional effects. In MQgel@Mito-treated rat hearts, the upregulation of genes encoding respiratory chain components (Mt-nd4, Cox4i1, Ndufa8, Atp5me) and contractile proteins (Tpm1, Actc1, Myh6), coupled with downregulation of inflammatory (Il3ra, Il1rap) and apoptotic (Casp3, Bcl2l11) genes, aligned with the improved energy metabolism, enhanced contractility, attenuated inflammation, and reduced cell death.

Despite promising results, several limitations exist in the current study. First, although we demonstrated that MQgel extended mitochondrial viability, the optimal storage duration and conditions for clinical implementation require further systematic optimization. Second, the source of donor mitochondria, autologous versus allogeneic, remains to be addressed. While allogeneic mitochondria could enable off-the-shelf therapy, potential immune considerations warrant investigation. Third, MTR labeling, although widely employed for in vivo mitochondrial tracking, has inherent limitations: because the dye covalently binds mitochondrial proteins, residual fluorescence from degraded organelles may overestimate the presence of functional mitochondria [44]. Advanced technologies capable of reliably distinguishing exogenous from endogenous mitochondria post-transplantation are needed to fully elucidate the in vivo fate, distribution, and integration of transplanted mitochondria. Finally, to further dissect the therapeutic mechanism of MTT in post-MI treatment, macrophage-specific depletion models, single-cell sequencing, and lineage-tracing approaches are warranted to delineate cell-type-specific contributions and resolve the relative importance of macrophage-mediated immunomodulation.