SERCA is critical to control the Bowditch effect in the heart

Abstract

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca²⁺ in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCA^A617T induced positive staircase and arrhythmia, while SERCA^E442K accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect.

Figure 1

D. melanogaster exhibits negative staircase. (a) Semi intact preparation of a fly harboring the reporter system handC-GFP. (b) An amplified fluorescence image showing the fluorescent cardiomyocytes and pericardial cells. The arrow indicates the cell displacement trajectory. (c) Raw (above) and digitized (below) cell movements. (d) Semi intact preparation of a fly harboring the reporter system GCaMP3. (e) Images of intracellular Ca²⁺ transients tracked in the conical chamber. The recording represents an arrhythmic heart. (f) Amplified Ca²⁺ transient to indicate the different parameters analyzed. (g) Representative recording of mechanical activity showing lateral displacements of a single cell after three successive 0.5 Hz increments in stimulation frequency (ΔFr1, ΔFr2, ΔFr3) from basal (B) spontaneous heart rate. Right panel: twitches obtained after digitalization of images using an algorithm based in phyton language. (h) Left panel: representative line scans of calcium transient after three successive 0.5 Hz increments in stimulation frequency. Right panel: Digitized images of the respective line scans. (i) Quantification of mechanical activity (black symbols) and Ca²⁺ transients (white symbols), reveals the negative staircase in D. melanogaster. N = 20, 19, 13, 5 for mechanical activity data at basal, ΔFr1, ΔFr2, and Δ Fr3 respectively. N = 10, 10, 7, 4 for Ca²⁺ transients data at basal, ΔFr1, ΔFr2, and ΔFr3 respectively. (j) D. melanogaster exhibit significant FDAR only at the highest frequency. Quantification of half relaxation time of cell movement and Ca²⁺ transients. N = 11, 10, 5, 4 for mechanical activity data at basal, ΔFr1, ΔFr2, and ΔFr3 respectively. N = 9, 9, 8, 4 for Ca²⁺ transients data at basal, ΔFr1, ΔFr2, and ΔFr3 respectively. Two way ANOVA followed by Tukey’s test for multiple comparisons was used for statistical analysis.*, ^#P < 0.05.

Figure 2

SERCA mutations modify the staircase pattern in Drosophila melanogaster. (a) Mutant SERCA^A617T animals exhibit positive staircase and mutant SERCA^E442K animals negative staircase, compared to control individuals. (b) The time of half to relaxation did not change with an increment stimulation frequency in all transgenic lines. Amplitude and t_1/2: Control: N = 20, 1, 9. SERCA^A617T N = 8, 10, 8. SERCA^E442K N = 6, 5, 4. Two way ANOVA followed by Tukey’s test for multiple comparisons was used for statistical analysis. *P < 0.05 (between strains only in basal stimulation), ^#P < 0.05 (between strains in different frequency stimulation), & P < 0.05 (between strains). (c–e) A subgroup of flies were previously exposed (or not) to heat shock (closed and open symbols, respectively). The results are expressed as percentage of change related to the basal cardiac frequency. Data represent mean values ± S.E., and significance was calculated by two-way ANOVA, followed by Tukey’s post hoc test (a P value < 0.05 was considered significant). Unlike wild type counterparts, both mutants did not resist a third increment of frequency. For this reason, panel C presents three increments of frequency over the basal heart rate and panels d and e shows two increments of frequency. CONTROL: Heated: N = 20, 19, 13, 5. Not heated: N = 20, 17, 14, 4. SERCA^A617T: Heated: N = 8, 10, 8. Not heated: N = 9, 9, 3. SERCA^E442K: Heated: N = 6, 5, 4. Not heated: N = 6, 5, 3 at basal, ΔFr1, ΔFr2, and ΔFr3 (only for CONTROL) respectively.

Figure 3

The SERCA^A617T mutation induces arrhythmia. (a,b) Typical recordings comparing two individuals with regular and irregular heart rate (a,b). Top: wall displacement, middle panel: each line indicates one event ( = one beat), bottom: periods (intervals of time between maximum of two consecutive peaks) are more variable in (b,c) Raw data and average results (inset) of arrhythmicity index. Mutant SERCA^A617T animals exhibit significantly increased heart rate variability at basal heart rate and at the maximal frequency of stimulation. (d) Increased proportion of animal mutant for SERCA^A617T exhibit incremented ectopic beats at basal rates and at the maximal frequency of stimulation. Arrhythmia: Control: N = 29, 25, 2. SERCA^A617T N = 9, 8, 9. SERCA^E442K N = 4, 4, 3. Two way ANOVA followed by Tukey’s test for multiple comparisons was used for statistical analysis. *P < 0.05 (between strains only in basal stimulation), ^#P < 0.05 (between strains at different frequency stimulation), & P < 0.05 (between strains).