Disruption of sarcoendoplasmic reticulum calcium ATPase function in Drosophila leads to cardiac dysfunction

Abstract

Abnormal sarcoendoplasmic reticulum Calcium ATPase (SERCA) function has been associated with poor cardiac function in humans. While modifiers of SERCA function have been identified and studied using animal models, further investigation has been limited by the absence of a model system that is amenable to large-scale genetic screens. Drosophila melanogaster is an ideal model system for the investigation of SERCA function due to the significant homology to human SERCA and the availability of versatile genetic screening tools. To further the use of Drosophila as a model for examining the role of SERCA in cardiac function, we examined cardiac function in adult flies. Using optical coherence tomography (OCT) imaging in awake, adult Drosophila, we have been able to characterize cardiac chamber dimensions in flies with disrupted in Drosophila SERCA (CaP60A). We found that the best studied CaP60A mutant, the conditional paralytic mutant CaP60A(kum170), develops marked bradycardia and chamber enlargement that is closely linked to the onset of paralysis and dependent on extra cardiac CaP60A. In contrast to prior work, we show that disruption of CaP60A in a cardiac specific manner results in cardiac dilation and dysfunction rather than alteration in heart rate. In addition, the co-expression of a calcium release channel mutation with CaP60A (kum170) is sufficient to rescue the cardiac phenotype but not paralysis. Finally, we show that CaP60A overexpression is able to rescue cardiac function in a model of Drosophila cardiac dysfunction similar to what is observed in mammals. Thus, we present a cardiac phenotype associated with Drosophila SERCA dysfunction that would serve as additional phenotyping for further large-scale genetic screens for novel modifiers of SERCA function.

Figure 1. CaP60A ^kum170 mutant has altered heart rate and cardiac dimensions after heat shock.

A. Percent paralysis after heat shock. CaP60A ^kum170 heterozygote flies were exposed to heat shock of varying durations (no heat shock defined as 0 min (closed circle), 5 min (closed triangles), 7 min (open triangles), or 10 min (open circles)) and observed for up to 72 hours. All flies receiving a heat shock of greater than 7 minutes developed irreversible paralysis; while a smaller percentage of flies developed paralysis after 5-minute heat shock. B. Representative optical coherence tomography (OCT) recordings from w ¹¹¹⁸ and CaP60A ^kum170 after varying durations of heat shock (HS). End diastolic dimension (EDD) and end systolic dimension (ESD) are denoted in red; A 125 micron standard and one-second bar are shown. C. Heart rates measured from 3 second OCT recordings show a progressive decline in heart rate with increasing durations of heat shock. D. End diastolic dimensions (EDDs) increase with increasing duration of heat shock. E. Fractional shortening is not markedly altered with heat shock in the CaP60A^kum170 mutants. *p<0.05, † p<0.005, ‡p<0.0001 in comparison to 0 minutes heat shock by one-way ANOVA with Tukey’s multiple comparisons test. N= 16, 12, 16, 4, 22 for 0, 5, 6, 7 and 10 minute groups, respectively.

Figure 2. The effects of cardiac specific overexpression of CaP60A ^kum170 and wild type CaP60A on cardiac phenotypes.

A. Cardiac specific overexpression of wild type CaP60A (tinC–wtCaP60A) or mutant CaP60A^kum170 (tinC-CaP60A^kum170) in Drosophila is not sufficient to phenocopy the paralysis phenotype of the global CaP60A ^kum170 mutant. Each mutant had two copies of the transgene; B. Representative OCT images from transgenic CaP60A overexpression flies in comparison to w ¹¹¹⁸ and global CaP60A ^kum170. 125 micron standard and one-second bars are shown. C. Heart rate, D. End diastolic dimensions (EDD), E. Fractional shortening (%) in w ¹¹¹⁸, tinC–wtCaP60A and tinC-CaP60A ^kum170 and CaP60A ^kum170 at baseline and after 10-minute heat shock. No significant differences were noted in the heart rate in either tinC–wtCaP60A or tinC-CaP60A ^kum170 at baseline and after heat shock compared to w ¹¹¹⁸. *p<0.05, **p<0.005 vs. w ¹¹¹⁸ no heat shock, #p<0.05, # # p<0.005 vs. w ¹¹¹⁸ with heat shock, † p<0.0001 vs. w ¹¹¹⁸ with heat shock, tinC-CaP60A ^kum170 with heat shock, tinC-CaP60A ^kum170 with heat shock and CaP60A ^kum170 no heat shock by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 3. The effects of Calcium Release mutations on the CaP60A ^kum170 cardiac phenotypes.

A. Heat shock induced paralysis in heterozygous CaP60A ^kum170 flies as well as in transheterozygous mutants carrying both CaP60A ^kum170 and a mutation in Ryanodine receptor (Rya-r44F¹⁶). No heat shock induced paralysis was noted in the single Rya-r44F ¹⁶ mutants or w ¹¹¹⁸. B. Representative OCT images from w ¹¹¹⁸, CaP60A ^kum170, Rya-r44F ¹⁶, CaP60A ^kum170; Rya-r44F ¹⁶ with and without 10-minute heat shock; A 125 micron standard and one-second bar are shown. EDD= End diastolic dimension, ESD=End systolic dimension. C. Heart rate, D. EDD and E. Fractional Shortening (%) in w ¹¹¹⁸, CaP60A ^kum170, Rya-r44F ¹⁶, CaP60A ^kum170 ; Rya-r44F ¹⁶ at baseline and after 10-minute heat shock. *p<0.0002 vs. non-heat shock state of the same genotype, † p<0.0001 vs. CaP60A ^kum170 after heat shock, #p<0.0001 vs. w ¹¹¹⁸ of the same treatment condition, ‡p<0.0002 vs. Rya-r44F ¹⁶ of the same treatment condition by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 4. The effects of loss of CaP60A function on cardiac parameters.

A. Heat shock did not induce paralysis in flies with cardiac specific CaP60A RNAi (tinC>CaP60A RNAi), heterozygous deletion of CaP60A (Df(2R) BSC601), heterozygous p-element disruption of CaP60A (CaP60A^KG00570), or w ¹¹¹⁸ compared to CaP60A ^kum170. B. Representative OCT images in w ¹¹¹⁸, CaP60A ^kum170 after 10 minute heat shock, tinC>CaP60A RNAi, Df (2R) BSC601 and CaP60A ^KG00570; A 125 micron standard and one second bar are shown, C. Heart rate, D. End Diastolic Dimensions (EDD), E. End Systolic Dimensions (ESD) and F. Fractional shortening (%) in w ¹¹¹⁸, CaP60A ^kum170 after 10 minute heat shock, tinC>CaP60A RNAi, Df(2R) BSC601 and CaP60A ^KG00570. †p<0.001 vs. w ¹¹¹⁸, *p<0.05 vs. tinC>CaP60A RNAi, ‡p<0.0001 vs. w ¹¹¹⁸, **p<0.005 vs. w ¹¹¹⁸ by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 5. Rescue of Cardiac function with CaP60A overexpression.

A. Representative OCT images of w ¹¹¹⁸, heldup ² (hdp²) and cardiac specific CaP60A (tinC-wtCaP60A) overexpression in hdp ² genetic background (hdp²; tinC-wtCaP60A); Scale bar= 125µm, EDD= End diastolic dimension, ESD=End systolic dimension. B. Heart rate, C. EDD (µm), D. ESD (µm) and E. Fractional shortening (%) in w ¹¹¹⁸, CaP60A ^kum170 after 10 minute heat shock, tinC>CaP60A RNAi, Df(2R) BSC601 and CaP60A ^KG00570. †p<0.001 vs. w ¹¹¹⁸, *p<0.05 vs. tinC>CaP60A RNAi, ‡p<0.0001 vs. w ¹¹¹⁸, **p<0.005 vs. w ¹¹¹⁸ by one-way ANOVA with Tukey’s multiple comparisons test.