Chronic cold exposure causes left ventricular hypertrophy that appears to be physiological

ABSTRACT

Exposure to winter cold causes an increase in energy demands to meet the challenge of thermoregulation. In small rodents, this increase in cardiac output leads to a profound cardiac hypertrophy, 2–3 times that typically seen with exercise training. The nature of this hypertrophy and its relevance to winter mortality remains unclear. Our goal was to characterize cold-induced cardiac hypertrophy and to assess its similarity to either exercise-induced (physiological) hypertrophy or the pathological hypertrophy of hypertension. We hypothesized that cold-induced hypertrophy will most closely resemble exercise-induced hypertrophy, but be another unique pathway for physiological cardiac growth. We found that cold-induced hypertrophy was largely reversed after a return to warm temperatures. Further, metabolic rates were elevated while gene expression and mitochondrial enzyme activities indicative of pathology were absent. A gene expression panel comparing hearts of exercised and cold-exposed mice further suggests that these activities are similar, although not identical. In conclusion, we found that chronic cold led to a phenotype that most closely resembled physiological hypertrophy, with enhanced metabolic rate, without induction of fetal genes, but with decreased expression of genes associated with fatty acid oxidation, suggesting that heart failure is not a cause of winter mortality in small rodents and identifying a novel approach for the study of cardiac growth.

Summary: Winter survival requires cardiac growth to supply elevated metabolism. Data in mice support physiological growth following chronic cold exposure, identifying an understudied form of hypertrophy.

INTRODUCTION

When faced with a functional challenge, the heart is capable of undergoing considerable hypertrophy to increase capacity (Snelling and Seymour, 2024, this issue). The challenge can be due to increased afterload (e.g. pressure overload or hypertension; Weeks and McMullen, 2011) which necessitates increased force production to maintain cardiac output, or the heart may need to increase output (e.g. exercise or volume overload) in response to increased metabolic demand (Dorn, 2007; Lavie et al., 2015; Nakamura and Sadoshima, 2018). In both cases, increased ventricular mass and wall thickness facilitates increased contractility (Dorn, 2007). While the cardiac hypertrophy appears similar, pressure overload commonly progresses from wall thickening to an enlarged organ with thinning walls; this dilated cardiomyopathy ultimately leads to failure, as this response is usually not reversible (Grossman et al., 1975; Katz, 1991; Weeks and McMullen, 2011). Typically, with volume overload, when an individual increases cardiac output, hypertrophy of the myocardium occurs without pathological outcomes and is reversible upon a return to normal levels of demand (Dorn, 2007); this ‘physiological hypertrophy’ occurs in response to exercise (athlete's heart; Dorn, 2007; Rawlins et al., 2009) as well as pregnancy (Schannwell et al., 2002; Ritterhoff and Tian, 2023).

While most work on cardiac hypertrophy has been done on these models, cardiac hypertrophy has also been shown following prolonged cold exposure in several rodent species including guinea pig (Kayar and Banchero, 1985), rats (Schechtman et al., 1991; Cheng and Hauton, 2008) and mice (Mineo et al., 2012), as well as in the gray short-tailed opossum (Schaeffer et al., 2003). Given that thermoregulation is a core homeostatic response, winter cold must be tolerated via sufficient heat production in mammals and birds that remain active year-round at high latitudes or altitudes. While shivering or non-shivering thermogenesis may occur in multiple tissues (Nowack et al., 2017), it is ultimately the heart that must supply heat-producing tissues with oxygen- and nutrient-rich blood, resulting in a continuous volume overload. As such, this approach can provide valuable proof-of principle for the relevance of continuous cardiac activity in driving hypertrophy. Work to date describing the cardiac phenotype following chronic cold exposure includes greater capillarity to maintain diffusion distances (Kayar and Banchero, 1985), increased lipid uptake (Cheng and Hauton, 2008) and partial reversal after a return to warm conditions (Schechtman et al., 1991), all of which are consistent with physiological hypertrophy. Indirect evidence supporting this includes the increase in metabolic capacity seen in many species after winter acclimation (Schaeffer et al., 2001, 2003; Mineo et al., 2012; Sgueo et al., 2012; Swanson and Vézina, 2015; Hayward et al., 2022). Despite these consistencies with a physiological phenotype, recent reviews (see Portes et al., 2023; Kong et al., 2020) posit that cold exposure is pathological, as other work has shown that cold exposure leads to hypertension. Rats (Sun et al., 1997b) and mice (Sun et al., 2003a,b) exposed to chronic cold develop both cardiac hypertrophy and hypertension, suggesting that cold may drive a pathological pressure-overload hypertrophy. However, the effects of cold exposure on blood pressure were eliminated by inhibition of the renin–angiotensin system (Sun et al., 1997b, 2003a,b) or by inhibition of beta-adrenergic stimulation (Sun et al., 1997a), although neither manipulation inhibited cardiac hypertrophy. Thus, the nature of cardiac hypertrophy in response to chronic cold exposure remains unclear. The application of insights gained from the study of cold-induced cardiac hypertrophy to heart failure will require understanding of the nature of this phenotype.

Heart failure due to cardiac hypertrophy has obvious human health effects, and heart disease remains a primary health risk worldwide (Vaduganathan et al., 2022). There is a pressing need to continue to clarify the gene regulatory networks instructing pathological and physiological hypertrophy to formulate interventions capable of preserving or restoring cardiac function. Knowledge of the physiological pathways associated with other disease states has led to successful disease treatment options, including the use of beta blockers for hypertension (Ram, 2010; do Vale et al., 2019), and janus kinase inhibitors in blocking the downstream response to cytokine production in autoimmunity such as arthritis or vitiligo (Banerjee et al., 2017; Qi et al., 2021). Understanding the underlying mechanisms driving distinct cardiac hypertrophy phenotypes may similarly reveal avenues for treatment, especially in models that match the chronic nature of stresses that lead to pathological hypertrophy. As a natural model of cardiac hypertrophy, the regulatory pathways stimulated by cold exposure offer an opportunity to achieve this goal. Regardless of whether cold exposure causes a physiological or pathological form of hypertrophy, the result is an integrated organismal response to a common environmental challenge and thus likely representative of an evolved adaptation. Several gene suites differ between pathological and physiological hypertrophy including major differences in biomechanical and metabolic function (Nakamura and Sadoshima, 2018; Ritterhoff and Tian, 2023). During development, the heart switches from a fetal to an adult gene expression program. This program includes switching of myosin isoforms as well as upregulation of transcriptional regulators of fatty acid metabolism, as the adult heart relies primarily on fatty acid fuels (Neely et al., 1972). The hallmark of pathological hypertrophy is the reversion to expression of the fetal gene program (Nakamura and Sadoshima, 2018; Taegtmeyer et al., 2010). One aspect of this reversion is switching to greater reliance on glucose metabolism and a reduction of fatty acid oxidation (FAO; Huss and Kelly, 2005). Genetic ablation of metabolic genes in mice (e.g. Schaeffer et al., 2004; Tolwani et al., 2005), as well as human data from inborn errors of metabolism (Vockley, 2008; Olpin, 2013) make it clear that metabolic abnormality can act as the root cause of heart failure.

In physiological hypertrophy, a reactivation of the fetal gene expression program following exercise (Dorn, 2007) is not observed. There is evidence for enhanced metabolic capacity with physiological hypertrophy (Abel and Doenst, 2011). Determining whether a model represents a physiological or pathologic phenotype has, until recently, relied on observing a lack of pathology (Dorn, 2007). Several commonalities between pregnancy- and exercise-induced physiological hypertrophy have since been established, including increased angiogenesis, cell survival and proliferation – although the exact molecular pathways used to achieve this are not fully established (Nakamura and Sadoshima, 2018). Despite these similarities, exercise and pregnancy are not identical. For example, pregnancy differs from exercise in its continuous nature, in the hormonal environment and in some of the molecular pathways used (Chung and Leinwand, 2014). However, the key element of any physiological phenotype includes a return to normal after the return to a non-stressful environment (Dorn, 2007). Our goal in this study was to determine the nature of cold-induced cardiac hypertrophy (CICH) in mice, and the extent to which it resembles pathological or physiological hypertrophy. While available data based on organismal performance following cold exposure (Mineo et al., 2012; Cheng and Hauton, 2008; Schaeffer et al., 2003) suggest to us that cold exposure leads to a physiological hypertrophy, they are insufficient to firmly support that conclusion. Exercise is the most common model for physiological hypertrophy, but it differs from other models in being periodic rather than continuous. Exposure to chronic cold is continuous but this modality is under-explored and it remains unclear what form of hypertrophy occurs. Two alternative hypotheses present themselves. First, chronic cold exposure in mice is pathological as a result of the continuous demand and presence of hypertension. If true, we would predict that markers of pathology (such as switching to the fetal gene program, lack of reversibility, fibrosis and reduced metabolic capacity) would be present following winter conditions. Second, the heart undergoes physiological hypertrophic remodeling in order to sustain increased demands, and is able to tolerate this stress. If this second hypothesis is supported, cold-exposed mice would return to normal left ventricular mass after a return to normal temperatures, and we would also expect a lack of fetal gene induction during cold exposure – instead, we would expect the induction of oxidative metabolism. We would also expect, but not necessarily require for support of this hypothesis, that cold-induced cardiac hypertrophy would exhibit similar hypertrophy markers to other models of physiological hypertrophy.

MATERIALS AND METHODS

Animals

We used male lab mice (Mus musculus Linnaeus 1758) for three experiments and deer mice [Peromyscus leucopus (Rafinesque 1818)] and meadow voles [Microtus pennsylvanicus (Ord 1815)] for another experiment. For the first experiment, we captured wild deer mice and meadow voles at Miami University's Ecology Research Center (ERC) near Oxford, OH, USA (39°30′N, 84°44′W) during both summer (June 2018) and winter (February 2019). Voles and deer mice were caught in the ERC's 1 hectare restored prairie using Sherman and wire drop-door traps with grain placed inside the traps as bait. Extra precautions were taken in winter to prevent death by hypothermia by leaving bedding in traps and not setting traps during precipitation and below-freezing temperatures. As soon as deer mice or voles were trapped, they were brought to the laboratory for tissue collection.

For the second experiment, we used FVB wild-type mice from a colony maintained at Miami University's Laboratory of Animal Resources (LAR). For the third experiment, we purchased C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME, USA) and maintained them in the LAR as described below. Experiment four used C57BL/6J mice from a colony maintained at Miami University's LAR. After weaning, or upon arrival, all mice were single housed in standard cages and light cycles with water and standard rodent diet (Lab Diet 5001, Cincinnati Lab Supply Inc., Cincinnati, OH, USA) provided ad libitum. Except for cold exposure mice (see ‘Experimental design’, experiments 2, 3 and 4, below), the room temperature was set at 23°C and humidity was 30–50%.

In all cases, after experimental procedures or wild capture, animals were euthanized with inhaled CO₂ followed by cervical dislocation. Tissues collected are noted in each experimental design section. All animal experimentation was approved by the Institutional Animal Care and Use Committee of Miami University (protocols 717, 718, 805, 919 and 993) and complied with the ‘Principles of Animal Care’, publication no. 86-23, revised 1985, of the National Institutes of Health as well as the laws of the USA.

Experimental design

Experiment 1

To characterize the cardiac response to naturally occurring cold exposure, we captured 9–10 individuals of both meadow voles and deer mice in winter or summer (10 voles and 10 deer mice per group, except 9 deer mice in summer). We collected body mass and tissue mass for the whole heart, left ventricle (LV), liver, spleen, gastrocnemius and kidney. The sex of individuals was mostly male with some females and a few for which sex was not identified.

Experiment 2

To begin to assess the nature of cold-induced cardiac hypertrophy, at 4 weeks of age, we placed 21 mice into either a control or cold-exposure group. For the first 2 weeks, cold-exposure mice were kept in incubators (Model I41VL Controlled Environment Chamber, Percival Scientific Inc., Perry, IA, USA) while the temperature was incrementally lowered from 23°C to 4°C (∼1.5°C per day). After 4°C was reached, we kept the cold-exposure mice at 4°C for another 7 weeks. At 13 weeks of age, we euthanized all mice and collected body mass, LV mass and froze LV samples to determine metabolic capacity (via a citrate synthase assay, see below) or for quantification of expression of selected target genes (via qPCR, see below).

Experiment 3

To compare cold exposure with treadmill running exercise, 18 mice, 6 weeks old, were randomly placed into a control, cold-exposure or treadmill running group. Mice in the cold-exposed group were kept in incubators (Model I41VL Controlled Environment Chamber, Percival Scientific Inc.) for 2 weeks while the temperature was incrementally lowered as in experiment 2. After 4°C was reached, we kept the cold-exposure mice at 4°C for another 6 weeks. Beginning at the same age as cold exposure, we ran the treadmill group on an EXER 3R treadmill (Columbus Instruments, Columbus, OH, USA) for 8 weeks. Similar to the cold-exposure group, we ramped up the intensity of running for the initial 2 weeks, starting at 10 m min⁻¹ for 12 min and ending at 25 m min⁻¹ for 45 min after 2 weeks. These mice then ran at 25 m min⁻¹ for 45 min, 5 days a week for the following 6 weeks. In all cases, mice ran up a 10 deg incline. Three days before the end of the experimental manipulation, we measured the rate of oxygen consumption over a 24 h period (described below). After the 8 weeks of cold exposure or treadmill running, all mice were euthanized. We measured body mass and LV mass, and stored LV tissue for Next Generation sequencing (see below).

Experiment 4

We used 46 male mice to determine the reversibility of cold-induced cardiac hypertrophy. Mice were randomly assigned to control or cold-exposure groups. Six-week old mice in the cold-exposed group were kept in incubators (Model I41VL Controlled Environment Chamber, Percival Scientific Inc.) for 2 weeks while the temperature was incrementally lowered as in experiment 2. After 4°C was reached, we kept the cold-exposure mice at 4°C for another 8 weeks, as described in experiment 2. In addition to sampling after the 8 weeks of cold exposure, we returned some cold-exposed individuals to standard conditions and, together with their age-matched control, collected tissues after 4, 5 or 8 weeks of ‘recovery’ at room temperature. From cold-exposed and control animals, we collected body mass and LV mass from 9 pairs of mice immediately after cold exposure, 3 pairs after 4 weeks, 3 pairs after 5 weeks and 9 cold-exposed and 8 controls after 8 weeks of recovery.

Citrate synthase measurements

We measured citrate synthase (CS) activity in cardiac tissue from 5 cold-exposed and 5 control mice from experiment 2. We diluted the frozen samples 1:20 in a homogenization medium (50 mmol l⁻¹ Tris, 0.15 mol l⁻¹ KCl, pH 7.4), homogenized them in a Dounce glass homogenizer and centrifuged homogenates at 1000 g at 4°C for 10 min. The resultant supernatant was frozen in liquid nitrogen and stored at −80°C. Maximal CS activity was determined spectrophotometrically at 25°C by measuring the rate of disappearance of acetyl-CoA at 232 nm in assay reagent (100 mmol l⁻¹ Tris, pH 8.1) with excess acetyl CoA (0.2 mmol l⁻¹) and oxaloacetate (0.17 mmol l⁻¹) over a 4 min interval.

Metabolic rate measurements

We measured daily metabolic rate 3 days before the end of experiment 3 in 6 control, 6 cold-exposed and 5 exercised mice via indirect calorimetry using a Physioscan System (AccuScan Instruments, Inc., Columbus, OH, USA). Individual mice were weighed, then placed in a sealed metabolic chamber (volume of ∼1 l). Constant flow through the chamber was maintained using a mass flow meter controlled by a flow controller (0.5 l min⁻¹). Excurrent gas passed through a Drierite column to remove water, then the O₂ and CO₂ analyzers. Flow rate and gas composition were collected at a rate of one sample per second. Data were calculated as oxygen consumption rate per whole animal and per gram body mass.

We measured individuals at their housing temperature; thus, controls and exercised mice were both measured at 23°C while cold-exposed mice were measured at 4°C. Each trial began at about 18:00 h and ran until 08:00 h a full day later (∼38 h). Data were analyzed over a 24 h period beginning at 20:00 h to allow the animals to quiet down in the cages. Data were separated into light phase or dark phase and a mean value for each period was determined for each mouse.

qPCR

RNA was extracted from the LVs of 11 mice (6 control, 5 cold exposed) from experiment 2 using TRIzol^® Reagent (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentrations were determined using a NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA) and diluted to a final concentration of 0.1 μg μl⁻¹ with nuclease-free water. A total of 1 μg RNA was then reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions on a PTC-100 thermocycler (Bio-Rad). cDNA was then used as a template for quantitative real-time PCR (qPCR). Each PCR reaction mixture was composed of cDNA template, 0.1 or 0.05 μmol l⁻¹ gene-specific primers (primer-specific optimal concentration) and 2× iQ SYBR^® Green Supermix (Bio-Rad) in a total of 15 μl. The temperature cycles for each qPCR reaction were as follows: 3 min at 95°C, 40 cycles of 95°C for 12 s, and a primer-specific optimal temperature (55–63.7°C) for 45 s. Each qPCR run was completed with a melt curve analysis to confirm the presence of a single PCR product and amplification efficiency was verified for every primer pair. Expression levels were determined using the ΔΔCt method.

Next generation sequencing/TRex

We surveyed the literature for genes reported to be altered in either pathological or physiological hypertrophy and assessed expression using Next Generation Sequencing. After dissection, total RNA was extracted from LVs of mice from experiment 4 using TRIzol^® Reagent (Ambion). RNA concentrations were verified using a NanoDrop (Nanodrop Technologies). Library preparation was completed using TruSeq Targeted RNA Custom Kit (cat. no. RT-101-1001, Illumina) as specified by TruSeq Targeted RNA Expression Reference Guide (15034665, Illumina) and libraries were prepared using unique oligonucleotides for our 160 candidate genes (Table S1). TE buffer (5 µl) was used for the hybridizing oligos step, and sequencing was run on the Illumina MiSeq Benchtop NGS platform. Analysis was conducted through the The BaseSpace^® TruSeq^® Targeted RNA v.1.0.1 application provided by Illumina Base Space (https://www.illumina.com/). Alignment, and the quantification of the relative expression of genes and abundance across samples were all conducted via the TruSeq^® Targeted RNA app. This application relies on v.2.5.57.4.TREx of the Isis analysis software; v.0.1.19-isis-1.0.3 of SAMtools; v.0.14.0 of Scipy; and v.0.14.1 of Pandas for annotations. Over 1,800,000 reads for each condition were aligned using a Burrows Wheeler aligner to align the sequences. A total of 95.8% of reads were aligned for the 5 cold-exposed mice and 95.9% were aligned for both the control and the exercised mice. The normalized read counts between conditions had an R²=0.98 for exercised versus control, R²=0.97 for cold-exposed versus control, and R²=0.99 for cold-exposed to exercised.

Analysis and statistics

To compare relative LV mass, we used a one-way ANCOVA with body mass as a covariate to look at the effect of treatment (cold exposure and exercise, or summer and winter). To compare metabolic rate measurements (V̇_O2), we used a two-way ANOVA to look at the effects of treatment (cold exposure and exercise) and time of day (day and night). To compare CS measurements, we used a t-test to determine the effect of cold exposure activity. To examine recovery from cold exposure, we used a two-way repeated measures ANCOVA with body mass as a covariate to determine the effect of treatment (cold exposure versus control) and time on LV mass. The qPCR efficiencies were validated for each gene, and expression levels were normalized to that of the 36B4 (acidic ribosomal phosphoprotein P0) housekeeping gene, which did not change with treatment. A two-tailed t-test assuming equal variances was used to determine the differential expression of the normalized qPCR results between cold and control individuals. For the TREx data, differential gene expression was calculated between each group using DeSeq2 through the Amplicon App on Illumina Base Space (https://basespace.illumina.com/). All tests were conducted with α=0.05 significance level in R v.4.2.3.

RESULTS

We first determined whether LV mass from animals living in the cold was higher than that of controls. We saw no clear gross morphological differences between the LVs in any of the conditions. There were significant effects of winter on relative LV mass in wild-caught meadow voles and deer mice (Fig. 1). For deer mice, relative LV mass was 50.4% larger in winter than in summer (P<0.0001), while for meadow voles, relative LV mass was 22.3% larger in winter than in summer (P<0.01). These results indicate that LV hypertrophy is a component of winter survival in wild rodents, likely due to the elevated energetic demands of thermoregulation. Additionally, we saw that the right ventricle (RV) was 14.2% larger in winter in meadow voles, although this was not statistically significant (P=0.07). The RV was 46.8% larger in deer mice (P≤0.001; Fig. S1). Hypertrophy was only seen in the hearts, as the liver, kidney, spleen and gastrocnemius muscle did not differ across season for either species (Fig. S1). As previously reported (Mineo et al., 2012), hypertrophy was also seen in laboratory mice after prolonged cold exposure (Fig. 1), with mean relative LV mass 30.2% greater than in control mice (P<0.0001). These results indicate that 7 weeks of cold exposure similarly generated sufficient energetic demand to induce cardiac hypertrophy in lab mice.

Fig. 1.

Relative left ventricle (LV) mass was significantly higher in winter in deer mice and meadow voles, and higher after prolonged cold exposure in lab mice. Values are means±s.e.m. (deer mouse summer n=10, winter n=9; meadow vole summer n=10, winter n=10; lab mouse control n=5, cold exposed n=9); asterisks indicate significant pairwise differences in LV mass between groups with body mass as a covariate (*P<0.05).

We then compared the hypertrophy following cold exposure with that following endurance exercise training. The effects of cold exposure and exercise were significant (ANCOVA: F_3,13=28.51, P<0.0001). Pairwise comparisons showed that relative LV mass was 38.8% larger in cold-exposed mice than in control mice (Fig. 2; P<0.0001), while relative LV mass was 11.3% larger, although not significantly so, in exercised mice than in control mice (P=0.182). We also saw that the relative LV mass was 25% larger in cold-exposed mice than in exercised mice (P<0.0001). These results suggest that 6 weeks of cold exposure was a stronger stimulus for cardiac hypertrophy than daily exercise over the same period.

Fig. 2.

LV mass was significantly higher in cold-exposed mice than in both control and exercised mice. Values are means±s.e.m. (control n=6, cold exposed n=6, exercised n=5); asterisks indicate significant pairwise differences between groups with body mass as a covariate (****P<0.0001).

We asked whether CICH was reversible. After prolonged cold exposure, we found that hypertrophy was significantly reduced following a return to standard conditions (F_2,43=30.28, P<0.0001). Similar to Figs 1 and 2, immediately after cold exposure, pairwise comparisons revealed that relative LV mass was 30.4% larger than in control mice (Fig. 3; P<0.0001). After a 4 week recovery period, mean relative LV mass was 23% larger than in control mice (P<0.0066), but significantly lower than immediately after cold exposure (P<0.01). While relative LV mass continued to decline after the return to standard conditions, there was no significant difference between relative LV mass in the cold-exposed mice 4 weeks into the recovery period and onward (pairwise comparisons for 4 and 5 weeks, P=0.242; for 4 and 8 weeks, P=0.253; and for 5 and 8 weeks, P=0.998). Further, after 8 total weeks of recovery, the relative LV mass was still 10.1% larger than that of control mice (P<0.05), although significantly lower than immediately after cold exposure (P=0.001). These results indicate that while cardiac hypertrophy is significantly reduced after removal from the cold, some is retained for at least 8 weeks.

Fig. 3.

Relative LV mass in mice was significantly higher following 8 weeks of cold exposure and returned to near control values, but remained significantly higher than controls after 4, 5 and 8 weeks of recovery in standard temperatures. Values are means±s.e.m. (week 0 end of cold exposure n=9, control n=9; week 4 post-cold exposure n=3, control n=3; week 5 post-cold exposure n=3, control n=3; week 8 post-cold exposure n=9, control n=7). Different lowercase letters indicate significant pairwise differences of LV mass within and between groups with body mass as a covariate.

To examine cardiac function, we used oxygen consumption (V̇_O2) as an indirect measure of the animals’ capacity to elevate cardiac output. Metabolic rate (MR) was significantly elevated following 6 weeks of cold exposure (Fig. 4), during both day and night phases (F_3,30=554.5, P<0.0001). Pairwise comparisons revealed that cold-exposed mice had a significantly higher resting MR than control mice, with the mean MR in cold-exposed mice being 2.3-fold higher than that of control mice during the day (P<0.0001) and 2.2-fold higher than that of control mice during the night (P<0.0001). There were no significant differences in resting MR between exercise-trained and control mice during the day or night. These results demonstrate the intensity of the energetic demand experienced during cold exposure and the capacity of mice to maintain continuous activity.

Fig. 4.

Sustained metabolic rate (MR) during cold exposure was more than 2-fold higher than that measured in standard conditions, in both daytime inactive and nocturnal active phases. Exercise training had no effect on 24 h resting MR. Values are means±s.e.m.; different lowercase letters indicate significant differences between groups (control n=6, cold exposed n=6, exercise n=5).

We next asked whether the oxidative capacity of the LV was altered after cold exposure using CS activity as a marker of mitochondrial capacity. There were no significant effects of cold exposure on LV CS activity (P=0.598; Fig. 5); thus, prolonged cold exposure had no effect on LV oxidative capacity.

Fig. 5.

Citrate synthase (CS) activity in LV muscle in 14 week old mice following 8 weeks of cold exposure was not significantly altered. Values are means±s.e.m. (control n=5, cold exposed n=5).

To test for induction of pathological signaling, we quantified gene expression of 12 genes, all of which are differentially regulated in models of pathological hypertrophy. Of these, only one was differentially expressed between cold-exposed and control mice (Table 1). Expression of brain natriuretic peptide (NPPB) was significantly downregulated in cold-exposed mice compared with controls, opposite to its response to pathological hypertrophy and heart failure. Thus, downregulation with cold exposure, in conjunction with the absence of significant differential expression in the other 11 known pathology marker genes, indicates an absence of a signal for pathological hypertrophy in the cold-exposed mice compared with controls. While incomplete, this lack of clear pathology prompted further investigation into other less commonly used markers of pathology, and general gene expression differences between cold-exposure and exercise training, a known model of physiological hypertrophy.

Table 1.

Differential mRNA expression of known hypertrophy marker genes suggests a lack of pathological hypertrophy in cold-exposed mice

To begin to understand the molecular signaling pathways involved in cold-induced hypertrophy, we examined the differential expression of 160 genes in the LVs of control, cold-exposed and exercise-trained mice using TruSeq Targeted RNA sequencing (TREx). Some of the targeted genes have a known association with pathological or physiological hypertrophy, and others were examined to obtain an overview of the transcriptomic signature of the cold-exposed heart in well-known cardiac genes. For these 160 genes (Table S2), we conducted three pairwise comparisons of the differential gene expression: control versus cold exposed (7 genes differentially expressed), control versus exercised (10 genes differentially expressed), and cold-exposed versus exercised (6 genes differentially expressed). In total, only 16 genes were differentially expressed across all groups, and half of these genes were found to be differentially expressed in multiple comparisons (Table 2).

Table 2.

TREx panel supports a physiological hypertrophy following cold exposure

In the first comparison, between control and cold-exposed mice, four out of the seven differentially expressed genes were downregulated (Table 2, Fig. 6A). Both DIABLO and MYH7 were both significantly downregulated; their upregulation would be a sign of pathological hypertrophy. The downregulation of PPAR gamma coactivator 1 alpha (PPARGC1a) and IRS2 may indicate pathology. Of the three genes that were upregulated, sarcolipin (SLN) and glucokinase (GCK) are considered a physiological response when upregulated. Matrix metallopeptidase 3 (MMP3), is more complicated as its upregulation is often associated with pathology. However, MMP3 has also been associated with physiological models of hypertrophy such as pregnancy and exercise (Chung et al., 2012).

Fig. 6.

Heatmaps of pairwise significant differential gene expression. Data are for the 16 genes showing differential expression out of the 160 targeted genes from the LVs of cold-exposed (n=5), control (n=6) and exercised (n=4) mice from z-scores of the normalized count data. Seven candidate genes had significantly different expression between cold-exposed mice (Cold) and controls (Con; A), 10 genes significantly differed in expression between exercised mice (Ex) and controls (B), and six genes significantly differed in expression between cold-exposed and exercised mice (C) (P≤0.05). Many of the known markers of pathology among the 160 targeted genes were not differentially expressed in any of the comparisons. Expression patterns between cold-exposed mice and exercised mice partially overlap when compared with control (SLN and MMP3). Meanwhile, several other genes show the opposite pattern in the two conditions of hypertrophy, such as SLC27A1, which is upregulated in exercised mice but downregulated in cold-exposed mice, and MYH7, which is downregulated in the cold-exposed mice but unchanged in the exercised mice. The similarity of the gene expression patterns overall suggests that cold exposure drives a different response from exercise training, but that it is more closely related to physiological than to pathological hypertrophy.

Of the 10 genes differentially expressed in the exercised compared with the control mice (Fig. 6B), three were upregulated and seven downregulated. SLN and MMP3 were upregulated, similar to that seen in the cold-exposed versus control comparison, as well as NADH:ubiquinone oxidoreductase core subunit V1 (NDUFV1). As with the cold-exposed mice, both SLN and MMP3 are likely signs of physiological hypertrophy and cardiac remodeling, respectively. The upregulation of NDUFV1 is consistent with physiological hypertrophy as it is a mitochondrial respiratory gene. However, the seven genes that were downregulated included cytochrome c1 (CYC1), which is another mitochondrial respiratory gene, PPARGC1a as well as PPARG coactivator 1 beta (PPARGC1b). As these are coactivators of mitochondrial oxidation and fatty acid oxidation, we would expect upregulation of the latter two in a physiological model. Their downregulation may indicate pathology or, as induced factors, that their response had already returned to baseline after 7 weeks of chronic exposure. The remaining downregulated genes, actin alpha cardiac muscle 1 (ACTC1), NK2 homeobox 5 (NKX2-5) and pyruvate dehydrogenase kinase 4 (PDK4), may show upregulation or downregulation with pathological hypertrophy, depending on the models.

When we compared the cold-exposed mice with the exercised mice directly (Table 2, Fig. 6C), we found six genes that showed significantly different expression between the two conditions. Of particular interest, MYH7 was downregulated in the cold-exposed mice compared with exercised mice, similar to when compared with controls. Upregulation of SLC27A1 in exercised mice when compared with cold-exposed mice is in concordance with increased FAO usage and was only seen in the exercised mice. While not significantly different when comparing the cold-exposed and exercised groups, the gene expression of PPARGC1a was downregulated in both experimental groups compared with the control animals. PPARGC1b expression was also downregulated in both groups compared with controls, but only significantly so in the exercise versus control comparison. Likewise, SLN and MMP3 were upregulated in both groups compared with control animals, but were not significantly different from one another.

In addition to the findings above, several known markers of pathology were not differentially expressed for any comparison. These include (but are not limited to), myosin light chain 2 (MYL2), myosin light chain kinase 2 (MYLK2), ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 2 (ATP2A2), three different caspase-2 isoforms (1, 3 and 8), heat shock protein family B (small) member 2 (HSPB2), heat shock protein 90 alpha family class B member 1 (HSP90AB1) and heat shock protein family D (Hsp60) Member 1 (HSPD1) (Table S2). These results are largely congruent with an absence of molecular indicators of pathology in the cold-exposed heart.

DISCUSSION

Cardiac hypertrophy is most simply divided into pathological and physiological (Dorn, 2007; Nakamura and Sadoshima, 2018). Pathological hypertrophy, best known in response to hypertension, is characterized by compensatory LV wall thickening followed by subsequent thinning and progressive loss of function with a lack of reversibility, which ultimately leads to failure if the cause is not removed (Bazgir et al., 2023). Physiological hypertrophy, often called the athlete's heart, is also characterized by enlargement of the LV, but occurs without loss of function or progression to failure, and typically reverses upon cessation of the growth stimulus (Bazgir et al., 2023; Pittaras et al., 2023). In addition to the many gene manipulations that lead to cardiac hypertrophy, the causes of hypertrophy can also be broken down into pressure overload or volume overload. Pressure overload is typically associated with pathology while volume overload is often associated with physiological hypertrophy. Either schema oversimplifies these categories, failing to capture the range of available responses (Pitoulis and Terracciano, 2020). For example, pregnancy and exercise training both cause volume overload and are considered physiological hypertrophy, but differ in many ways (Chung et al., 2012). As our model of chronic cold exposure has some apparent overlap with both exercise (increased metabolic demand) and pregnancy (continuous demand), we predicted that chronic cold exposure would lead to a physiological CICH. The enhancement of whole-animal metabolic capacity in cold-exposed or winter-acclimatized animals (e.g. Schaeffer et al., 2001; Mineo et al., 2012) and the observation that many mammals living through winter conditions survive for several annual cycles supports that this is a healthy, physiological response. Opposing these points is the correlation between cold exposure and hypertension in humans (Rios et al., 2023; Rose, 1961) as well as the increased risk of adverse cardiac events in winter (Liu et al., 2015). Additionally, work in rodents has shown that chronic cold exposure also causes cold-induced hypertension (CIH; Shechtman et al., 1991; Sun et al., 1995, 1997a,b, 2003a,b; Sun, 2010; Chen and Sun, 2017). Importantly, mechanistic studies have repeatedly shown that CIH and CICH are independent of one another (Shechtman et al., 1991; Sun et al., 1995, 1997a,b, 2003a,b, 2005; Sun, 2010; Chen and Sun, 2017). Other models of physiological hypertrophy, such as pregnancy, have co-occurrence with hypertension as is the case in gestational hypertension (de Haas et al., 2017). Nonetheless, winter mortality is relatively high in many small mammals (Gliwicz and Taylor, 2002; Boonstra and Krebs, 2012). We are unaware of any study that has investigated the long-term effects of overwintering over several annual cycles on cardiac structure or function; thus, contributions of cardiac damage due to winter cold on mortality are also unexplored.

While a clear definition of physiological hypertrophy is lacking, regression to normal size after removal of the stressor may be the most compelling characteristic (Bazgir et al., 2023; Pittaras et al., 2023). Similar to other models of physiological hypertrophy, our male mice also showed a nearly complete regression to control heart mass. Most of this occurred by 4 weeks and it is important to note that complete regression had not occurred by 8 weeks after removal from the cold, similar to what was observed previously (Shechtman et al., 1990). We also found that animals maintained functional capacity, as animals sustained elevated metabolic rates throughout cold exposure. The continuous energetic demand during thermoregulation may explain why the degree of cardiac hypertrophy seen after cold exposure (∼30%) exceeds that seen in most exercise models (∼10–15%). While we did not see evidence for increased fatty acid oxidation or mitochondrial oxidation after cold exposure, nor were these downregulated, this is similar to what is commonly observed following exercise (Dorn, 2007). The structural and metabolic phenotype we observed support that cold-induced cardiac hypertrophy is a form of physiological hypertrophy. More measures such as echocardiography and cardiac catheterization are warranted to better characterize the functional phenotype.

Gene expression data predominately support a pattern of physiology. The qPCR results showed a significant decrease in NPPB expression (Table 1), while increased expression levels of NPPB, a member of the fetal gene program, have been associated with heart failure (Dirkx et al., 2013). None of the genes that would be expected to be upregulated in a pathological phenotype, such as ANP, MYH7 and NKX2-5, were upregulated and one of the genes, NPPB, showed the opposite result of what we would expect in pathology. Further, the FAO genes whose gene expression was measured in the targeted qPCR showed no change as a result of cold exposure, when we would have expected those to be upregulated if cold exposure resembled exercise-induced hypertrophy. Similarly, for the TREx gene expression panel, many of the significantly differentially expressed genes support a pattern of physiological hypertrophy (Fig. 6). Following cold exposure, the downregulation of DIABLO (a well-known apoptosis gene) and MYH7 (myosin heavy chain β) are both indicative of a physiological response. MYH7 is expressed in the heart in a ratio with the gene for α-myosin. A shift from its adult ∼90%:10% β/α expression ratio to an even higher proportion of β-myosin has historically been used as a marker of pathological hypertrophy (Nakao et al., 1997). Not only is the downregulation of β-myosin the opposite pattern we would expect in pathology but also β-myosin has been shown to decrease in hearts responding positively to beta-blockers (Lowes et al., 2002). Upregulation of both SLN and GCK is considered a physiological response. The increased expression of SLN has been implicated as a possible pathological marker; however, it likely acts as a method for increasing energy supplies to the cardiomyocytes through Ca²⁺ modulation (Bal and Periasamy, 2020). The increased expression of both SLN and MMP3 in both our cold-exposed and exercised groups indicates that these genes may be more heavily related to changes in the extracellular matrix rather than pathology (Magdalena et al., 2006). There are also signs that the strain on the heart may contain elements of pathology. One example of this from our dataset is that we would expect an increase in FAO in physiological hypertrophy, as was seen in the exercised mice. Our exercised animals showed an upregulation of some FAO-associated genes (Fig. 6C) such as Slc27a1, while our cold-exposed animals did not show a change in these when compared with control mice. This may be indicative of pathology in the cold-exposed mice as they do not appear to be upregulating lipid metabolism to meet their energetic demands. Many forms of pathological hypertrophy are accompanied by a downregulation of mitochondrial respiratory genes (Abel and Doenst, 2011), as well as the PPAR/PGC family of regulatory proteins (Huss and Kelly, 2005). Physiological hypertrophy leads to upregulation of several members of this family (Baar, 2004; Ehrenborg and Krook, 2009; Muoio and Koves, 2007; Vega et al., 2017). The decrease in expression of both PPARGC1a and PPARGC1b in our cold-exposed mice would typically be considered a marker of pathology. However, PPARGC1b expression was also significantly decreased in our exercised mice, which was unexpected. PPARGC1a was also decreased when compared with controls, but not significantly so. Our cold-exposure model matching our exercised-induced (physiological) model supports that cold exposure also resulted in physiological hypertrophy. One possible explanation for this pattern is that our time course missed the uptick in expression by these co-activators, as they have been shown to have fast-acting and transient induction (Baar et al., 2004).

Although many signaling pathways regulating physiological hypertrophy are known (Nakamura and Sadoshima, 2018), physiological hypertrophy can be described as a lack of pathology (Dorn, 2007). As the signaling pathways between exercise and pregnancy are not identical (Chung and Leinwand, 2014), it stands to reason that CICH may use unique molecular pathways to achieve a physiological phenotype as well. Thus, the lack of change in known markers of pathology should be considered as support for a physiological phenotype. Additionally, some gene expression patterns may be indicative of either pathology or physiology depending on the model. One example of this context dependence that is relevant to our study is the regulation of PDK4 expression, the downregulation of which is associated with increased FAO, typically seen in physiological hypertrophy. However, the opposite pattern of regulation is seen in pregnancy, where PDK4 expression is upregulated as it is thought to inhibit pyruvate dehydrogenase and prevent pyruvate flux and thus FAO in late-stage pregnancy models (Liu et al., 2017). PDK4 expression matched published reports in our exercised animals, but did not change in the cold, which suggests that cold exposure may induce a novel set of molecular pathways to drive cardiac hypertrophy as it follows neither model. Clearly, we cannot determine on a gene-by-gene basis whether a model of hypertrophy is inherently pathological or not (Dorn, 2007), and must instead look at a mixture of markers and physiological endpoints. We measured many indicators of pathology that showed no change in expression in genes such as NKX2-5, GATA4 and MEF2 isoforms from the fetal gene program, common stress markers (such as heat-shock proteins) among others (Table S2). However, we did not look at several key components associated with cardiac function including blood pressure, cardiac output, fractional shortening or the presence of increased fibrosis. These, along with the relationship between CICH and CIH, require future study.

Combined with our structural and functional data, the overall gene expression pattern displayed in cold-induced cardiac hypertrophy appears to be most similar to a physiological model of hypertrophy, with some differences in the molecular pathways being employed when compared with exercise. While it is tempting to suggest that cold-induced hypertrophy must be physiological, as animals with extended lifespans survive multiple winters, there is simply insufficient data to draw such a conclusion, especially as winter mortality can be quite high and cardiac contribution to mortality in these cases is unexplored. Cold exposure has also been implicated in pathological hypertrophy when at extreme low temperatures (−20°C), showing LV dysfunction and oxidative stress in mouse models (Cong et al., 2018). Despite these observations, overwintering is a relatively stable stressor that organisms in cold climates must prepare for and live through each year. Our results indicate that mammals which thermoregulate through winter conditions undergo cardiac hypertrophy as an adaptation to increased thermal demands. Future work on the adaptive nature from an ecological evolutionary perspective should include female mice, as well as studies that incorporate realistic variability in winter temperatures or that span multiple annual cycles and that directly measure mortality as well as reproductive success following winter conditions. Even when physiological in nature, cardiac hypertrophy is a response to a stressor to improve cardiac function and meet demands. Over time, continuous demand may become a pathological stressor. When we consider the relationship with human health issues, pathways activated in a physiological schema may lead to feasible targets for treatment. The identification of such targets may be accomplished through a thorough comparison between this emerging model and the currently known models of hypertrophy. These include the physiological model of pregnancy which matches cold exposure in its continuous volume overload, obesity-induced hypertrophy and pressure overload, both of which are the most relevant human pathologies. Studies including full-transcriptome comparisons, investigations into small-RNA correlations and fuller functional analyses comparing across other hypertrophy models may begin to reveal answers to therapeutic problems.