Chamber-specific regulation of myofilament Ca2+ sensitivity by nNOSβ via troponin I phosphorylation: Predominance in the right ventricle

Figures

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  Figure 1.. Similar levels of total nNOS expression and NO production between LVCMs and RVCMs. (A) Representative immunoblots showing total nNOS, p-nNOS (p-nNOSSer1417), and GAPDH in LVCMs and RVCMs. (B) Quantification of total nNOS expression normalized to GAPDH in LVCMs (N = 10) and RVCMs (N = 10). (C) Quantification of p-nNOSSer1417 expression normalized to GAPDH in LVCMs and RVCMs. (D) Ratio of p-nNOSSer1417 to total nNOS in LVCMs and RVCMs. No significant differences were detected between groups. (E) Nitrite content measured in LVCMs and RVCMs under control conditions (C) and following inhibition of nNOS with L-NAME (L) or SMTC (S). Both inhibitors significantly reduced nitrite levels in LVCMs and RVCMs compared with control, with no significant difference between ventricular cell types (N = 9). Each dot represents one biological sample from an individual animal, and one sample per animal per condition was included in the quantitative analysis. n.s., not significant. Source data are available for this figure: SourceData F1.

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  Figure S1.. Differential phosphorylation of eNOS in LVCM and RVCM. (A) Representative immunohistochemistry showing nNOS staining in transverse sections of adult rat hearts. Low-magnification images indicate the regions of the LV and RV used for higher-magnification views. Enlarged images illustrate representative staining patterns in LVCM and RVCM (scale bar = 2 mm). (B) Representative immunoblots showing total eNOS, phosphorylated eNOS at Thr495 (p-eNOSThr495), and phosphorylated eNOS at Ser1177 (p-eNOSSer1177) in LVCM and RVCM. (C) Quantification of total eNOS expression. eNOS protein levels were significantly lower in RVCM compared with LVCM. (D) Quantification of p-eNOSThr495 expression. No significant difference was observed between LVCM and RVCM. (E) Quantification of p-eNOSSer1177 expression. Phosphorylation at Ser1177 was significantly increased in RVCM relative to LVCM. Each dot represents one biological sample from an individual animal, and one sample per animal per condition was included in the quantitative analysis (N = 16 animals). Source data are available for this figure: SourceData FS1.

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  Figure 2.. nNOS inhibition enhances sarcomere shortening and delays relaxation more prominently in RVCMs. (A) Representative SL traces from LVCMs and RVCMs under control conditions and following nNOS inhibition with SMTC. The timing of electrical stimulation and peak shortening are indicated. Solid black line, LVCM control; black dotted line, LVCM + SMTC; solid gray line, RVCM control; gray dotted line, RVCM + SMTC. The timing of electrical stimulation and peak shortening are indicated. (B) SL traces from panel A normalized to the amplitude of sarcomere shortening (ΔSL) to compare the time course of shortening and relaxation across conditions. (C) Diastolic SL measured in LVCMs and RVCMs under control (C) and SMTC-treated (S) conditions. (D) Absolute sarcomere shortening (ΔSL) in LVCMs and RVCMs under control and SMTC-treated conditions. (E) Sarcomere shortening normalized to diastolic SL (ΔSL/diastolic SL). (F–H) Time to 10% (F), 50% (G), and 90% (H) relengthening following peak sarcomere shortening in LVCMs and RVCMs under control and SMTC-treated conditions. In panels C–H, gray dots represent individual cardiomyocytes, whereas black dots represent the mean value for each animal. Sample…

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  Figure S2.. Distribution of sarcomere shortening and relaxation parameters in LVCM and RVCM with or without SMTC. (A) Distribution of diastolic SL. (B) Distribution of sarcomere shortening amplitude (ΔSL). (C) Distribution of fractional sarcomere shortening (ΔSL/diastolic SL). (D) Time to 10% relaxation. (E) Time to 50% relaxation. (F) Time to 90% relaxation. Data are shown as box-and-whisker plots representing cell-level measurements from LVCM, LVCM + SMTC, RVCM, and RVCM + SMTC. Boxes indicate the interquartile range with the median shown as a horizontal line; whiskers denote the minimum and maximum values. Each box represents the distribution of values obtained from individual cardiomyocytes pooled across animals.

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  Figure 3.. Effects of nNOS inhibition on Ca 2+ transients in LVCMs and RVCMs. (A) Representative intracellular Ca2+ transient traces recorded as F360/F380 in LVCMs under control conditions and following inhibition of nNOS with SMTC. Solid black line, LVCM; black dotted line, LVCM + SMTC. The timing of electrical stimulation and peak Ca2+ signal are indicated. (B) Representative Ca2+ transient traces from RVCMs under control conditions and following SMTC treatment. Solid gray line, RVCM; gray dotted line, RVCM + SMTC. (C) Resting Ca2+ signal expressed as F360/F380 was measured. (D) Peak Ca2+ signal (F360/F380 at peak) measured in each experimental group. (E) Ca2+ transient amplitude expressed as ΔF360/F380 at peak. (F–H) Time to 10% (F), 50% (G), and 90% (H) decay of the Ca2+ transient following peak. In panels C–H, gray dots represent individual cardiomyocytes, whereas black dots represent the mean value for each animal. Sample sizes were as follows (N = 10 animals per group): LVCM, n = 103; LVCM + SMTC, n = 201; RVCM, n = 127; RVCM + SMTC, n = 136. n.s., not significant.

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  Figure S3.. Cell-level distributions of Ca 2+ transient parameters in LVCM and RVCM with or without SMTC. (A) Resting Ca2+ level expressed as F360/F380 at rest. (B) Peak Ca2+ signal (F360/F380 at peak). (C) Ca2+ transient amplitude (F360/F380 at peak). (D) Time to 10% decay of the Ca2+ transient. (E) Time to 50% decay of the Ca2+ transient. (F) Time to 90% decay of the Ca2+ transient. Data are presented as box-and-whisker plots showing cell-level measurements from LVCM, LVCM + SMTC, RVCM, and RVCM + SMTC. Boxes represent the interquartile range with the median indicated by a horizontal line; whiskers denote the minimum and maximum values. Each box reflects the distribution of Ca2+ transient parameters obtained from individual cardiomyocytes pooled across animals.

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  Figure 4.. Effects of nNOS inhibition on L-type Ca 2+ channel current (ICa,L ) in LVCM and RVCM. (A) Representative ICa,L traces recorded from LVCM under control conditions and following SMTC treatment using the perforated whole-cell configuration. Solid black line, LVCM; black dotted line, LVCM + SMTC. Cells were held at −40 mV and depolarized to test potentials between −40 and +50 mV in 10 mV increments. (B) I-V relationships of peak ICa,L normalized to cell capacitance (pA/pF) in LVCM and LVCM + SMTC. Cell-level comparison of peak ICa,L density at the voltage producing maximal inward current demonstrated a statistically significant difference between groups (P < 0.0001). (C) Representative ICa,L traces recorded from RVCM under control conditions and following SMTC treatment using the same voltage protocol as in panel A. Solid gray line, RVCM; gray dotted line, RVCM + SMTC. (D) I-V relationships of peak ICa,L normalized to cell capacitance (pA/pF) in RVCM and RVCM + SMTC (P = 0.0008). Sample sizes were as follows: LVCM, n = 38 cells; LVCM + SMTC, n = 36 cells; RVCM, n = 38 cells; RVCM + SMTC, n = 32 cells.

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  Figure 5.. Ca 2+ -sarcomere length (SL) relationship in LVCM and RVCM with or without SMTC. (A) Cell-level phase-plane plots showing the relationship between sarcomere length and intracellular Ca2+ signal (F360/F380) in LVCM (solid line) and LVCM/SMTC (dotted line). Traces represent averaged trajectories constructed from individual cardiomyocytes across the full Ca2+ transient. (B) Corresponding Ca2+-SL relationships in RVCM (solid line) and RVCM/SMTC (dotted line).

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  Figure 6.. Effects of SMTC on cytosolic Ca 2+ buffering properties in LVCM and RVCM. (A) Ca2+-binding ratio (κ) plotted as a function of intracellular Ca2+ concentration ([Ca2+]c) in LVCM, LVCM + SMTC, RVCM, and RVCM + SMTC. Total buffering capacity (circles), intrinsic buffering (squares), and fura-2 buffering (triangles) are shown. Lines indicate fits derived from buffering analysis (Eq. 1 and Eq. 2). (B) Calculated total bound Ca2+ as a function of [Ca2+]c in LVCM (top) and RVCM (bottom). Solid lines indicate calculated values for control cells, and dashed lines indicate calculated values for SMTC-treated cells. Shaded areas represent the difference in bound Ca2+ between control and SMTC conditions across the measured Ca2+ range (<2 µM). (C) Summary of total endogenous Ca2+ buffer concentration ([S]tot) in LVCM and RVCM with (S) or without (C) SMTC. No significant differences were detected between groups. (D) Summary of intracellular fura-2 concentration ([fura-2]tot) across experimental groups. SMTC treatment did not affect indicator loading in either LVCM or RVCM. (E) Apparent dissociation constant (Kd,S) of endogenous Ca2+ buffers. SMTC significantly reduced Kd,S in both LVCM and…

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  Figure S4.. Representative experimental traces used for intracellular Ca 2+ buffer analysis. (A) Representative cytosolic Ca2+ concentration ([Ca2+]c) trace obtained from a control LVCM during sequential application of thapsigargin, caffeine, and a Na+- and Ca2+-free solution. After confirming Ca2+ transients evoked by depolarizing steps from −70 to 0 mV for 200 ms, SERCA was inhibited with thapsigargin while stimulation was continued. Caffeine (20 mM) was then applied to confirm depletion of SR Ca2+ stores. Finally, the normal Tyrode solution was replaced with 0Na+/0Ca2+ solution to inhibit NCX-mediated Ca2+ transport. (B) Representative [Ca2+]c response to a series of membrane depolarizations used to determine the relationship between Ca2+ influx and the corresponding increase in cytosolic Ca2+ concentration (Δ[Ca2+]c). Dots indicate 100-ms depolarizing pulses. After a steady state was reached, depolarizing pulses from −40 to 0 mV were applied at 1 Hz for 6–8 repetitions to evoke inward Ca2+ currents. (C) Representative inward Ca2+ currents evoked by the depolarizing pulses shown in B. (D) Time integrals of the inward Ca2+ currents, expressed as the quantity of electrical charge and used…

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  Figure 7.. SMTC treatment reduces TnI phosphorylation. (A) Representative immunoblots showing total troponin I (TnI), phosphorylated TnI at Ser23/24 (p-TnISer23/24), and α-actinin in LVCM and RVCM under control conditions (C) or following SMTC treatment (S). α-Actinin was used as a loading control. (B) Quantification of total TnI expression normalized to α-actinin. No significant differences were observed between control and SMTC-treated cells in either LVCM or RVCM. (C) Quantification of p-TnISer23/24 expression normalized to α-actinin. SMTC treatment significantly reduced p-TnI levels in RVCM but not in LVCM. (D) Ratio of p-TnISer23/24 to total TnI. SMTC induced a modest reduction in the p-TnI/TnI ratio in LVCM and a pronounced reduction in RVCM. Each dot represents one biological sample from an individual animal, and one sample per animal per condition was included in the quantitative analysis (N = 12 animals). Source data are available for this figure: SourceData F7.

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  Figure 8.. Enhanced nNOS phosphorylation in the myofilament fraction of RVCM compared with LVCM. (A) Representative immunoblots showing total nNOS, p-nNOS at Ser1417 (p-nNOSSer1417), and α-actinin in the myofilament fractions isolated from LVMF and RVMF. α-Actinin was used as a loading control. (B) Quantification of total nNOS expression normalized to α-actinin in LVMF and RVMF. No significant difference was observed between groups. (C) Quantification of p-nNOSSer1417 expression normalized to α-actinin. p-nNOSSer1417 levels were significantly higher in RVMF compared with LVMF. (D) Ratio of p-nNOSSer1417to total nNOS. The p-nNOS/nNOS ratio was markedly increased in RVMF relative to LVMF. Each dot represents one biological sample from an individual animal, and one sample per animal per condition was included in the quantitative analysis (N = 16 animals). Source data are available for this figure: SourceData F8.