Fig 2: LETM1 knockdown modulates Ca2+ efflux in permeabilized H9c2 cells. (A) mRNA expression of LETM1 in H9c2 cells transiently transfected with scrambled (siScr) or LETM1 siRNA (siLETM1) for 48 h, normalized to the GAPDH mRNA level. (B) Representative immunoblot and (C) densitometric quantification of LETM1 protein expression in H9c2 cells treated with scrambled or LETM1 siRNA for 48 h, with GAPDH as loading control. Bars in (A,C) represent mean ± SEM (n = 3) relative to scrambled siRNA. *p < 0.0005 versus. control siRNA. (D) Changes in cytosolic Ca2+ in permeabilized WT H9c2 cells (black) or H9c2 cells transfected with LETM1 siRNAs (red), in response to 5 µM CaCl2 boluses when energized with 10 mM K-Succinate. (E) Rate of change of Ca2+ calculated using linear fits for Ca2+ efflux rate in WT and 1-exponential fits for the decay rate in [Ca2+]c in siLETM1 H9c2 cells following the 4th CaCl2 bolus in (D). Further details provided in section “Materials and Methods.” (F) Changes in cytosolic Ca2+ in permeabilized WT H9c2 cells in response to a 5 µM CaCl2 bolus followed by addition of 1 µM Ru360 and 2 µM CGP at the times indicated by dashed lines.

Fig 3: The MRPP3 N434S variant reduces its association with LETM1 via MRPP2.(A) BioID of BirA*-tagged wt or N434S MRPP3 expressed in HeLa cells and the associated proteins were detected by mass spectrometry. Mitochondrial-targeted BirA* (mt-BirA*) was used as a control. (B) Average Venus intensities normalized to red fluorescent protein (RFP) intensity for interactions between LETM1 and the wt or N434S MRPP3, as well as MRPP1 and MRPP2 in NIH-3T3 cells determined by fluorescence-activated cell sorting. GST1 was used as a negative control. Data are shown as a percent change compared to the respective control. (C) Average Venus intensities normalized to RFP intensity comparing interactions between the wt and N434S MRPP3 with MRPP1 or MRPP2. GST1 was used as a negative control. (D) Immunoprecipitation of HA-tagged MRPP3 and MRPP3-N434S expressed in NIH-3T3 cells and immunoblots of the fractions from the immunoprecipitation against LETM1 and MRPP2. HA-tagged SOD2 was used as a control bait. (E) The levels of MRPP2 and LETM1 were measured by immunoblotting of pancreatic mitochondria and normalized to porin. Amino acid content was measured in pancreata (F) or serum (G) by mass spectrometry. All data are representative of results obtained from at least three experiments or three mice from 20-week-old N434S mice and wt mice fed an NCD or an HFD. Error bars are SEM *P < 0.05 and **P < 0.01, Student’s t test.

Fig 4: The MRPP3 N434S variant results in reduced mitochondrial calcium and insulin secretion.Cellular (A) and mitochondrial (B) calcium levels were analyzed in isolated pancreatic ß cells using Fura-2 or Rhod-2 fluorescent microscopy, respectively, under basal conditions and after exposure to glucose. (C) Potassium levels were measured in serum and (D) insulin levels were measured in pancreata by enzyme-linked immunosorbent assay (ELISA). (E) Insulin and glucagon levels were measured in pancreata by fluorescent immunohistochemistry. Hoechst staining was used to visualize the nuclei. (F) Glucose-stimulated insulin secretion was measured in pancreatic islet cells in the presence of low and high glucose, and total insulin levels were measured in the cell pellets as controls using an ELISA. All data are representative of results obtained from at least three mice from 20-week-old N434S mice and wt mice fed a NCD or a HFD. Error bars are SEM *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test. (G) Reduced RNase P association with LETM1 may affect calcium transport into mitochondria and the cellular calcium availability required for insulin secretion. The increased calorie intake from the HFD causes an increased requirement for insulin secretion, and the calcium levels are not sufficient to trigger increased insulin secretion that may eventually lead to insulin resistance.