Doxorubicin was purchased from Selleckchem (Houston, TX) in powder form (25 mg) and dissolved in sterile water as 1.25 mL aliquots to a concentration of 20 mg/mL and stored at 4°C. For injections, the 20 mg/mL stock solution was diluted in sterile saline to a final concentration of 1 mg/mL.

Animal protocols were performed according to NIH guidelines with approval from the Institutional Animal Care and Use Committee, Environmental Health and Occupational Safety Committee and the Biosafety Committee at Mayo Clinic. Mice were bred and maintained under pathogen-free conditions in the animal facility at the Mayo Clinic, fed standard chow and water ad libitum, and housed in animal rooms where the temperature was monitored. Breeding pairs of B6.129 wild-type (WT) (Cat#101045) and B6.129 Trpc6 whole body knock-out (KO) mice (26) (Cat#37345) were obtained from the Jackson Laboratory (Bar Harbor, ME). Male and female WT and Trpc6 KO mice (8–10 weeks old), ten mice per group, received either 100 μL intraperitoneally (ip) of control sterile saline or 4 mg/kg/dose doxorubicin for a cumulative dose of 24 mg/kg on days 1, 3, 5, 7, 9, 11 according to (20). Results were confirmed by repeating each experiment. Hearts were evaluated for cardiac function using echocardiography and tissues collected on day 14 and 21.

Cardiac function was performed by transthoracic echocardiogram using a Visual Sonic Vevo 2100 with a 55-megahertz (MHz) transducer (Bothell, WA). Echocardiography was performed on living male and female animals under isoflurane inhalation at day 14 and 21 as per our previous publications (20, 27–30).

Mouse hearts were cut longitudinally, fixed in 10% phosphate-buffered formalin, and embedded in paraffin for histological analysis. Five-micron-thick sections were stained with hematoxylin and eosin to detect vacuolation or trichrome blue to detect fibrosis. Vacuolation and fibrosis were calculated as the number of grids with vacuoles or fibrosis, respectively, compared to the total number of grids in the heart section using an eyepiece grid with a 2x objective lens (20x magnification) and converted to a percentage, as previously (31, 32). Sections were scored by two individuals blinded to experimental group.

At harvest, half of the heart was collected and stored at −80°C for RNA isolation. Hearts were homogenized and lysed using Tissuelyser (Qiagen) with 7 mm stainless steel beads in RTL buffer with 0.5% DX buffer to reduce foam (Hilden, Germany). The homogenate was then placed in an automated RNA isolation and purification instrument, QIAcube, with reagents for RNase Easy Fibrous Mini Kit including a DNase and Proteinase K step (Qiagen #74704). RNA was eluted into 30 μL. If the heart had been divided in the earlier step, the eluted RNA was pooled prior to being aliquoted. RNA quantification was determined in μg/μL using NanoDrop (Thermo Scientific, Waltham, MA).

Two-step quantitative reverse transcriptase-mediated real-time PCR (qPCR) was used to measure abundance of individual mRNAs. Total RNA from mouse hearts was assessed by quantitative real time (qRT)-PCR using Assay-on-Demand primers and probe sets and the ABI 7000 Taqman System from Applied Biosystems (Foster City, CA) after RNA was converted to cDNA using a High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems), and qPCR reactions were performed in triplicate with 100 ng of cDNA and the TaqMan Universal PCR master mix (Applied Biosystems), as previously described (28, 29). The following primer/probe sets were purchased from Applied Biosystems: Trpc1 (Mm00441975_m1), Trpc3 (Mm00444690_m1), Trpc6 (Mm01176083_m1), Myh7 (Mm00600555_m1), Tnni3 (Mm00437164_m1) and Rcan1 (Mm01213406_m1). Amplification data were collected with an Applied Biosystems ViiA7 detector and analyzed with ViiA7 v 1.2.4 software (Life Technologies). Data were normalized to the endogenous control Polr2a (Mm00839502_m1) (33) and mRNA abundance was calculated using the ΔΔCT method and displayed as fold change (FC) (34).

Hearts were fixed in 10% buffered formalin for 48 h and transferred to containers of PBS prior to paraffin embedding and mounting on slides. TUNEL Assay was performed using the Click-iT Plus TUNEL Assay for in situ Apoptosis Detection on the Alexa 647 (ThermoFisher, Cat: C10619). Slides were deparaffinized per manufacturer recommendations and steamed for 30 min prior to permeabilizing with Proteinase K. Tissue autofluorescence was quenched with Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Cat: SP-8400-15). Heart sections were incubated with TdT Reaction Buffer for 20 min at 37°C prior to performing the TdT Reaction for 60 min at 37°C. TUNEL reaction was performed for 45 min at 37°C. Nuclei were counter-stained with Hoechst 33342 (ThermoFisher, Cat: H21492) and then mounted with Vectashield Antifade Mounting Medium (Vector Labs, Cat: H-1000-10). After drying for 48 h, slides were scanned with a Panoramic 250 fluorescent slide scanner (3DHISTECH). The ventricles of heart sections were selected and annotated in CaseViewer (3DHISTECH). TUNEL positivity was determined in QuantCenter (3DHISTECH) using cell quant with the following parameters: Channel Matching – default; Detection – nuclei selected for both the DAPI and Cy5 channels; Nuclei – contrast set to 35, other settings were default; Cytoplasm – n/a; Membrane – n/a; Scoring – object selected was nuclei and channel selected was Cy5. These parameters allowed for identification of all nuclei and then determination of the frequency of TUNEL/Cy5 positivity where the aggregate score of Medium and Strong Positive Nuclei = TUNEL positive.

Statistical analyses were performed in GraphPad Prism 9.0.1. Differences between two groups were tested by unpaired 2-tailed Student's t-test. Differences between more than two groups were tested by one-way ANOVA followed by Tukey's or Holm-Sidak's multiple comparison tests. Differences between groups over time were compared by two-way ANOVA. Survival curves were analyzed by log-rank (Mantel-Cox) test. Data are expressed as mean ±SEM. A value of p < 0.05 was considered significant.

Shortly after the accumulative dose of doxorubicin was achieved at day 11 post inoculation, male wild-type and Trpc6-deficient mice began to die, although the specific cause of death was not ascertained (Figure 1). Deficiency in Trpc6 improved survival after doxorubicin treatment in males, (p = 0.003, Figure 1). These findings suggest that Trpc6 contributes to mortality following doxorubicin therapy in male mice.

Mice were weighed immediately prior to each injection of doxorubicin to ensure the correct dose was used (approximately 4mg/kg per dose). As expected, both wild-type and Trpc6-deficient males treated with doxorubicin progressively lost body-weight relative to control mice (p < 0.0001, Figure 2A), while wild-type and Trpc6-deficient control males maintained their weight over the duration of the experiment (p = 0.724, Figure 2A). The loss in weight for wild-type and Trpc6-deficient mice treated with doxorubicin was observed at day 21 (p < 0.0001, Figure 2B). However, Trpc6-deficient mice treated with doxorubicin lost less weight than wild-type mice treated with doxorubicin over the duration of the experiment (p < 0.0001, Figure 2A), suggesting that Trpc6 worsens the effects of doxorubicin.

In mice that survived to day 21, we also determined the heart-weight to tibia length (HW:TL) ratio. An elevated HW:TL indicates cardiac hypertrophy. Instead, we found that doxorubicin treatment caused a reduction in HW:TL in wild-type and Trpc6-deficient males (p < 0.001, Figure 2C), indicating cardiac damage, that was not recovered by Trpc6 deficiency (p = 0.64, Figure 2C). Thus, Trpc6 contributes to loss of body weight due to doxorubicin treatment but does not alter heart weight in male mice.

We next examined gene expression of two known biomarkers of heart damage, cardiac troponin (Tnni3) and myosin heavy chain 7 (Myh7, also known as myosin heavy chain beta), in male mice at day 21. Both Tnni3 and Myh7 gene expression was significantly different between groups by ANOVA (p < 0.0001 and p < 0.0001, respectively, Figures 3A,B). Tnni3 expression in the heart of wild-type mice was significantly reduced by doxorubicin treatment compared to saline controls, p < 0.0001, and the reduction was almost completely reversed by Trpc6 deficiency, (p < 0.0001, Figure 3A), indicating that Trpc6 promotes cardiac damage. Myh7 expression, which is known to increase in failing human (35, 36) and mouse hearts (37, 38), increased significantly in male wild-type mice treated with doxorubicin, p < 0.0001, and was also reversed by Trpc6 deficiency, (p < 0.0001, Figure 3B), indicating that Trpc6 promotes cardiac damage. The gene expression levels of Tnni3 and Myh7 were very similar between wild-type and Trpc6-deficient saline control males indicating that there was no apparent underlying difference in cardiac damage between the two mouse strains. Together, these data show that Trpc6 worsens cardiac damage in response to doxorubicin.

TUNEL Assay was performed to determine whether cardiac apoptosis was present 21 days after treatment with doxorubicin. We did not observe significant changes in apoptosis at day 21 after doxorubicin exposure between groups (Figure 3C). Fibrosis was found to be present in the heart at day 21 (Figure 4) and apoptosis is a process that primarily occurs prior to remodeling and fibrosis.

Vacuolation, a known effect of doxorubicin-induced cardiac damage in humans, was observed in male mice treated with doxorubicin (p < 0.0001, Figures 3D–H). Trpc6-deficiency significantly reduced cardiac vacuolation compared to wild-type controls following treatment with doxorubicin (p < 0.0001, Figures 3D–H), further demonstrating that Trpc6 promotes cardiac damage following doxorubicin treatment.

Cardiac fibrosis is well known to cause cardiomyopathy/dilated cardiomyopathy that can be detected by echocardiography in conditions such as viral myocarditis (31, 32). Cardiac fibrosis was assessed at day 21. Wild-type mice treated with doxorubicin showed a significant increase in fibrosis in the heart (p = 0.010, Figure 4A) while Trpc6-deficiency significantly decreased fibrosis (p = 0.028, Figure 4A).

Cardiac function was measured in male mice at day 14 and 21 by echocardiography (Figure 4). No significant changes were observed for any group at day 14 (data not shown). At day 21, wild-type mice treated with doxorubicin showed a significant decrease in heart rate, (p = 0.029, Figure 4B), LVEF, (p = 0.042, Figure 4C), fractional shortening, (p = 0.037, Figure 4D), cardiac output, (p < 0.0001, Figure 4E) and stroke volume, (p = 0.0001, Figure 4F) compared to wild-type control males. Trpc6-deficiency significantly improved cardiac function compared to wild-type males treated with doxorubicin for heart rate p = 0.022, LVEF p = 0.048, fractional shortening p = 0.043, cardiac output p = 0.002, and stroke volume p = 0.048, respectively (Figures 4B–F). Measures of left ventricular end diastolic and left ventricular end systolic diameters (LVEDD, LVESD) used to determine cardiac dilatation showed that neither doxorubicin nor Trpc6 deficiency led to dilated cardiomyopathy at this time point in males (Figures 4G,H). Thus, these data indicate that Trpc6 promotes cardiac damage that leads to cardiomyopathy following doxorubicin treatment in males.

The TRPC family of proteins (TRPC1-7) function as both homo- and hetero-tetramers, and both Trpc1 and Trpc3 as well as Trpc6 have been implicated in heart failure induced by pressure overload (23–25, 39). Another study reported that Trpc6 is a positive regulator of calcineurin-NFAT signaling through the regulator of calcineurin (Rcan1) (40). Therefore, we sought to characterize the changes in cardiac gene expression of Trpc6 in response to doxorubicin and Trpc1, 3 and Rcan1 in Trpc6-deficient mice after doxorubicin treatment.

In the hearts of male wild-type mice, we observed decreases in Trpc6, Trpc1 and Trpc3 gene expression in response to doxorubicin compared to saline controls, p = 0.0087, p = 0.032 and p < 0.0001, respectively (Figures 5A–C), but no significant change was observed in the expression of Rcan1 (Figure 5D). In Trpc6 deficient mice, the doxorubicin-induced changes in expression of Trpc1 and Trcp3 were reversed (Figures 5B,C). However, the expression of Trpc3 in the hearts of Trpc6 deficient control mice was significantly lower than that of wild-type control mice, (p = 0.004, Figure 5C), indicating that Trpc6-deficiency alters cardiac Trpc3 expression regardless of doxorubicin treatment.

Given that women with breast cancer are commonly treated with doxorubicin and that our initial genetic studies identified TRPC6 genetic variants as associated with a decline in LVEF in women with breast cancer (19), in this study we also assessed female mice treated with the same dose of doxorubicin as the dose given to males. In female wild-type mice, all wild-type and Trpc6-deficient mice survived treatment with doxorubicin (data not shown). In contrast to males, only Trpc6-deficient female mice treated with doxorubicin lost body weight over the duration of the experiment (Figure 6A). At day 21 (Figure 6B) wild-type female mice treated with doxorubicin maintained their weight, and no changes were observed in HW:TL in females for any group (Figure 6C).

As observed in male mice, Tnni3 cardiac gene expression was significantly reduced in wild-type females treated with doxorubicin (p = 0.050, Figure 7A), but unlike males, gene expression of Myh7 in wild-type females was not significantly altered by doxorubicin, (p > 0.999, Figure 7B). Similar to males, female mice developed vacuolation following treatment with doxorubicin, (p < 0.0001, Figure 7D) that was less severe than males (mean vacuolation in wild-type females treated with doxorubicin = 18.24% vs. 74.44% in males) (Figure 7D). And as with males, Trpc6-deficiency significantly reduced vacuolation in females treated with doxorubicin (p = 0.049, Figure 7D). Finally, we did not observe any significant change in cardiac fibrosis or echocardiographic parameters in female mice at day 21 in response to doxorubicin or Trpc6-deficiency (Figure 8). Thus, cardiac damage caused by doxorubicin was far less in females and did not lead to cardiomyopathy at day 21.

Although female wild-type and Trpc6-deficient mice were less susceptible to doxorubicin-induced cardiac damage and cardiomyopathy, we did observe other significant effects of Trpc6 deficiency in female mice compared to males.

In contrast to males (Figure 2A), female wild-type mice treated with doxorubicin did not lose weight (Figure 6A). The reason for this is not clear. Rather than wild-type mice being worse in males, Trpc6-deficient females treated with doxorubicin had a greater loss in body weight over time and at day 21 compared to wild-type mice treated with doxorubicin (p < 0.0001, Figures 6A,B). Although doxorubicin significantly decreased HW:TL (caused heart damage) in males (Figure 2C), there was no change in heart weight (no cardiac damage) in females (Figure 6C).

In contrast to males (Figure 3A), Tnni3 cardiac gene expression was significantly lower in Trpc6-deficient compared to wild-type saline control females (p < 0.0001, Figure 7A). In contrast to males, Tnni3 gene expression was significantly decreased in control and doxorubicin treated Trpc6-deficient females (Figure 7A), suggesting that Trpc6 deficiency altered Tnni3 levels in females. Pleiotropic effects of Trpc6 deficiency in response to doxorubicin were also observed for Myh7 gene expression in the hearts of female (Figure 7B) vs. male (Figure 3B) mice. In female mice, Myh7 levels remained low in all groups except for Trpc6-deficient mice treated with doxorubicin, where there was a significant increase relative to wild-type controls, (p = 0.049, Figure 7B).

In the hearts of female wild-type mice, doxorubicin induced a significant reduction in Trpc6 gene expression compared to wildtype controls (p = 0.039, Figure 9A) similar to the decrease observed in male mice (Figure 5A), but did not induce changes in Trpc1, Trpc3 or Rcan1 in wild-type mice (Figures 9B–D). A direct comparison of Trpc6 expression levels in the heart of male and female wild-type mice revealed that there were no significant differences in its expression before or after treatment with doxorubicin by sex (Figure 10). Interestingly, female Trpc6-deficient mice treated with saline had significantly lower expression of Trpc1, (p = 0.002, Figure 9B), Trpc3 (p < 0.0001, Figure 9C) and Rcan1 (p = 0.009, Figure 9D) than wild-type control mice. Thus overall, Trpc6 appears to increase cardiac damage in response to doxorubicin in females but not severely enough to lead to cardiomyopathy at the dose used in these experiments.