Myocardial ischemia/reperfusion (I/R) elicits an acute inflammatory response involving complement factors. Recently, we reported that myocardial necrosis was decreased in complement C3^−/− mice after heart I/R. The current study used the same heart model to test the effect of C3 on myocardial apoptosis and investigated if C3 regulation of apoptosis occurred in human cardiomyocytes. Comparative proteomics analyses found that cytochrome c was present in the myocardial C3 complex of WT mice following I/R. Incubation of exogenous human C3 reduced apoptosis in a cell culture system of human cardiomyocytes that did not inherently express C3. In addition, human C3 inhibited the intrinsic apoptosis pathway in a cell-free apoptosis system. Finally, human pro-C3 was found to bind with an apoptotic factor, pro-caspase 3, in a cell-free system. Thus, we present firsthand evidence showing that C3 readily reduces myocardial apoptosis via interaction with the intrinsic apoptotic pathway.

Plasma phospholipid transfer protein (PLTP) is a risk factor for cardiovascular diseases. Sphingosine-1-phosphate (S1P), carried by high-density lipoprotein (HDL), is a potent lipid mediator and is also associated with cardiovascular diseases. We found that germline Pltp gene knockout (KO) mice have decreased circulating S1P without influencing apoM, a major S1P carrier on HDL. We then hypothesized that, like apoM, PLTP is another S1P carrier. We established inducible Pltp-KO, Apom-KO, and Pltp/Apom double KO mice and measured plasma lipoprotein and S1P levels under different diets. We found that PLTP deficiency, and the double deficiency have a similar effect on HDL reduction. Importantly, we found that all mice have about 50% reduction in plasma S1P levels, compared to WT mice, and PLTP deficiency significantly reduces apoM levels (about 40%), while apoM deficiency has no effect on PLTP activity, indicating that PLTP depletion reduces S1P through HDL reduction. To further evaluate this HDL reduction-mediated effect, we overexpressed PLTP which also caused a reduction of HDL. We found that the overexpression reduces S1P and apoM as well as apoA-I, a major apolipoprotein on HDL. Furthermore, we found that albumin (another reported S1P carrier) deficiency in mice has no effect on plasma S1P. We also found that the influence of PLTP on HDL may not require its direct binding to the particle. In conclusion, PLTP is not a direct S1P carrier. PLTP depletion or overexpression in adulthood dramatically reduces plasma S1P through HDL reduction. ApoM, but not albumin, deficiency reduces plasma S1P levels.

Sphingomyelin synthase-related protein (SMSr) belongs to the SMS family; however, it cannot synthesize SM. We reported that SMSr is a phosphatidylethanolamine-specific phospholipase C, which is associated with metabolic dysfunction-associated fatty liver disease (MAFLD). However, the mechanism is unknown. Based on hierarchical clustering of the samples from the human Genotype-Tissue Expression project, we found that SMSr and serine palmitoyltransferase (SPT), the key enzyme for sphingolipid biosynthesis, as well as certain sphingolipid metabolism-related genes, belong to the same co-expression cluster in the liver and adipose tissues. We also found that Smsr expression is positively associated with Sptlc1 and Sptlc2 expression in both tissues of both genders. In a mouse study, we found that Smsr overexpression induced while Smsr knockout (KO) (under a high-fat diet) reduced SPT activity, thus, influencing most of the tested sphingolipids. Further, we found that PE treatment reversed Smsr overexpression-mediated SPTLC2 upregulation. PE supplement also reduced liver microsome SPT activity in a dose-dependent manner. Furthermore, we demonstrated that SMSr interacts with SPTLC2 in vivo. Thus, SMSr, as a member in the sphingolipid biosynthesis pathway, regulates SPT. Perturbation of SPT activity has been linked to the prevention of MAFLD and cardiovascular diseases. However, the approach to finding an SPT-specific inhibitor, as a drug, has not been successful so far. Importantly, global Smsr KO mice are viable and healthy; therefore, inhibiting SPT activity by reducing PE, mediated by SMSr/PE-PLC activity, could provide a novel approach for preventing and treating MAFLD.