Role of Protein Kinase D1-mediated NLRP3 Activation in Pathogenesis of Sepsis-induced Cardiomyopathy

M. Ghait (Greifswald)¹, L. Lang (Greifswald)¹, N. Dörmann (Greifswald)², J. Rüdebusch (Greifswald)¹, B. Brinschwitz (Greifswald)³, N. Li (Greifswald)¹, S. Utasi (Greifswald)², E. Martin (Greifswald)¹, P. Voori Giri (Greifswald)¹, K. Bartels (Greifswald)¹, B. Fielitz (Greifswald)¹, E. Hammer (Greifswald)⁴, U. Völker (Greifswald)⁴, J. Fielitz (Greifswald)^2
1University Medicine Greifswald Department of Internal Medicine B, Cardiology, DZHK (German Center for Cardiovascular Research), partner site Greifswald Greifswald, Deutschland; ²Universitätsmedizin Greifswald Klinik und Poliklinik für Innere Medizin B Greifswald, Deutschland; ³Universitätsmedizin Greifswald Klinik für Innere Medizin B Greifswald, Deutschland; ⁴Interfaculty Institute for Genetics and Functional Genomics Greifswald, Deutschland

Sepsis is a leading cause of mortality among critically ill patients and sepsis-induced cardiomyopathy (SICM) contributes to this outcome. Previously we reported that the NLRP3 inflammasome, a critical mediator of interleukin-1β (IL-1β) production and pyroptosis, plays a key role in SICM. NLRP3 is activated through phosphorylation by protein kinase D1 (PKD1) at the trans-Golgi network, which culminates in caspase-1 autoactivation. Active caspase-1 then processes IL-1β into its mature form and concurrently activates gasdermin-D, a membrane pore-forming protein, to facilitate IL-1β secretion and induce pyroptosis.
Here, we examined whether the PKD1-NLRP3 signaling axis is active in cardiomyocytes and if cardiomyocyte-specific deletion of PKD1 (Prkd1) could mitigate cardiac atrophy and maintain cardiac function during sepsis. Male cardiomyocyte-specific Prkd1 knockout (cKO; Prkd1^{loxP/loxP; αMHC-CRE}) mice and wild-type littermate controls (WT; Prkd1loxP/loxP) were subjected to polymicrobial sepsis through cecal ligation and puncture (CLP) surgery to facilitate SICM. Sham-operated mice served as controls. Cardiac function and atrophy were assessed 24 h post-surgery using transthoracic echocardiography. Neonatal rat ventricular cardiomyocytes (NRVCM) were used for mechanistic analyses. Immunoblotting and RT-qPCR analyses were used to measure protein levels in specific subcellular compartments and gene expression, respectively.
We observed that cKO mice were protected against SICM, as shown by less cardiac atrophy and preserved cardiac function compared to WT mice. Septic cKO mice showed a preserved left ventricular ejection fraction, fractional shortening, and decreased cardiac stress marker expression during sepsis compared to septic WT littermates. In vitro, NRVCMs showed robust NLRP3 activation and pyroptosis in a PKD1-dependent manner. Mechanistic studies revealed that PKD1 facilitated the relocation of NLRP3 from mitochondria to endomembrane compartments, essential for inflammasome activation. Collectively, these findings identify PKD1 as a critical modulator of SICM, orchestrating the spatiotemporal dynamics of NLRP3 inflammasome activity in cardiomyocytes.
Our results underscore the role of PKD1 as a pivotal mediator of SICM, influencing the spatiotemporal dynamics of the NLRP3 inflammasome within cardiomyocytes. This regulatory interaction between PKD1 and NLRP3 may be central to SICM pathogenesis.